Compositions And Methods For Epithelial Stem Cell Expansion And Culture

ABSTRACT

Described are cell culture solutions and systems for epithelial stem cell and organoid cultures, formation of epithelial constructs and uses of the same in transplantation.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/028,872, filed on Jul. 6, 2018, which is a continuation of U.S.application Ser. No. 14/775,560, filed on Sep. 11, 2015, now U.S. Pat.No. 10,041,046, which is the U.S. National Stage Application ofInternational Application No. PCT/US2014/023197, filed on Mar. 11, 2014,which designates the U.S., published in English, which claims thebenefit of U.S. Provisional Application No. 61/783,245, filed on Mar.14, 2013. The entire teachings of the above applications areincorporated herein by reference.

GOVERNMENT SUPPORT

This invention was made with Government support under Grant No. RO1DE013023 awarded by the National Institutes of Health. The Governmenthas certain rights to the invention.

INCORPORATION BY REFERENCE OF MATERIAL IN ASCII TEXT FILE

This application incorporates by reference the Sequence Listingcontained in the following ASCII text file:

a) File name: 00502283061_SEQUENCELISTING.TXT; created Mar. 16, 2021, 2KB in size.

BACKGROUND OF THE INVENTION

A single layer of epithelial cells that actively self-renews and isorganized into crypts and villi clothes the intestine. It has beenrecently shown that the renewal of intestinal epithelium is driven byLgr5⁺ intestinal stem cells (ISC) that reside at the base of thesecrypts (Barker et al., 2007). Lgr5⁺ stem cells can be isolated andcultured in vitro to form organoids containing crypt-villus structuresthat recapitulates the native intestinal epithelium (Sato et al., 2009).While these stem cells can be expanded for multiple passages in the formof organoids, existing culture conditions provide little to no controlover self-renewal and differentiation. Typical cultures consist ofheterogeneous cell populations, including stem cells and differentiatedcells (Sato et al., 2009). In particular, the self-renewal andproliferation of Lgr5+ stem cells both in vitro and in vivo aredependent on direct cell contact between Lgr5⁺ stem cells and anothercrypt cell type known as paneth cells (Snippert et al., 2010), whichsignificantly complicates and limits the ability to control the fate ofLgr5⁺ stem cells in culture. The inability to efficiently expand Lgr5⁺stem cells considerably limits the translation of this biology totherapies, where homogeneous stem cell cultures and efficient scale-upprocesses are essential prior to transplantation. Moreover, thereremains a need to develop improved, clinically-oriented systems fortransplantation of ex vivo expanded epithelial tissue into injuredrecipient organs.

SUMMARY OF THE INVENTION

In one aspect, the invention provides cell culture solutions.

In one embodiment, the invention provides a cell culture solutioncomprising an inhibitor of a Bone Morphogenic Protein, an inhibitor ofGlycogen synthase kinase-3 beta, an agent that binds to the Leucine-richrepeat-containing G-protein coupled receptor 5 and a histone deacetylaseinhibitor. In one embodiment, the inhibitor of Glycogen synthasekinase-3 beta can be CHIR99021, the agent that binds to the Leucine-richrepeat-containing G-protein coupled receptor 5 can be R-spondin 1, andthe HDAC inhibitor can be Valproic acid.

In another embodiment, the invention provides a cell culture solutioncomprising an inhibitor of a Bone Morphogenic Protein, at least about 3uM CHIR99021 and a histone deacetylase inhibitor.

In yet another embodiment, the invention provides a cell culturesolution comprising an inhibitor of a Bone Morphogenic Protein, an agentthat binds to the Leucine-rich repeat-containing G-protein coupledreceptor 5, a Wnt agonist and a HDAC6 inhibitor.

In yet another embodiment, the invention provides a cell culturesolution comprising an inhibitor of a Bone Morphogenic Protein,R-spondin 1, lithium chloride and a histone deacetylase inhibitor.

In yet another embodiment, the invention provides a histone deacetylaseinhibitor that is a Pan-HDAC inhibitor. The Pan-HDAC inhibitor can beselected from the group consisting of Valproic acid, Trichostatin A,suberoylanilide hydroxamic acid and Suberohydroxamic acid (SBHA).

In yet another embodiment, the invention provides a histone deacetylaseinhibitor that is an HDAC6 inhibitor. The HDAC6 inhibitor can beselected from the group consisting of Tubacin, Tubastatin A and Compound7.

In yet another embodiment, the invention provides an inhibitor of a BoneMorphogenic Protein that can be selected from the group consisting ofNoggin, Chordin, Follistatin, DAN, proteins comprising a DANcysteine-knot domain, Sclerostin, Twisted Gastrulation, UterineSensitivity-Associated Gene-1, Connective-Tissue Growth Factor, Inhibin,BMP-3 and Dorsomorphin.

In yet another embodiment, the invention provides an inhibitor ofGlycogen synthase kinase-3 beta that can be selected from the groupconsisting of CHIR99021, LiCl, BIO-acetoxime, CHIR98014, SB 216763, SB415286, 3F8, Kenpaullone, 1-Azakenpaullone, TC-G 24, TCS 2002, AR-A014418, TCS 21311, TWS 119, BIO-acetoxime, 10Z-Hymenialdisine, GSK-3βInhibitor II, GSK-3β Inhibitor I, GSK-3β Inhibitor XXVII, GSK-3βInhibitor XXVI, FRATtide peptide, Cdk1/5 Inhibitor and Bikinin.

In yet another embodiment, the invention provides an agent that binds tothe Leucine-rich repeat-containing G-protein coupled receptor 5 that canbe selected from the group consisting of R-spondin 1, R-spondin 2,R-spondin 3 and R-spondin 4.

In yet another embodiment, the invention provides a Wnt agonist that canbe selected from the group consisting of: Wnt-1/Int-1, Wnt-2/Irp(Int-I-related Protein), Wnt-2b/13, Wnt-3/Int-4, Wnt-3a, Wnt-4, Wnt-5a,Wnt-5b, Wnt-6, Wnt-7a, Wnt-7b, Wnt-8a/8d, Wnt-8b, Wnt-9a/14,Wnt-9b/14b/15, Wnt-10a, Wnt-10b/12, Wnt-11, Wnt-16, R-spondin 1,R-spondin 2, R-spondin 3, R-spondin 4, Norrin, CHIR99021, LiCl, BIO((2′Z,3′E)-6-Bromoindirubin-3′-oxime), CHIR98014, SB 216763, SB 415286,3F8, Kenpaullone, 1-Azakenpaullone, TC-G 24, TCS 2002, AR-A 014418,2-amino-4-[3,4-(methylenedioxy)benzyl-amino]-6-(3-methoxyphenyl)pyrimidine,IQ 1, DCA, QS 11, WAY-316606, (hetero)arylpyrimidines,10Z-Hymenialdisine, TCS 21311, TWS 119, GSK-3 Inhibitor IX, GSK-3Inhibitor IV, GSK-3β Inhibitor II, GSK-3β Inhibitor I, GSK-3β InhibitorXXVII, GSK-3beta Inhibitor XXVI, FRATtide, Cdk1/5 Inhibitor, Bikinin,and 1-Azakenpaullone.

In yet another embodiment, the invention provides a cell culturesolution comprising Noggin, R-spondin 1, CHIR99021 and an Atoh1inhibitor. The Atoh1 inhibitor can be an inhibitory nucleic acid.

In yet another embodiment, the invention provides a cell culturesolution further comprising an Epidermal Growth Factor and/or a Notchagonist. The Notch agonist can be selected from the group consisting ofa Notch1 antibody (N1 Ab), Delta 1, Delta-like 3, Delta-like 4, Jagged1, Jagged 2, DSL peptide and Delta D.

In yet another embodiment, the invention provides between about 5 toabout 500 ng/ml EGF, about 5 to about 500 ng/ml Noggin, about 50 toabout 1000 ng/ml R-spondin, about 0.1 to about 10 μM CHIR99021 and about0.1 to about 5 mM Valproic acid.

In yet another aspect, the invention provides cell culture systemscomprising cell culture solutions of the invention.

In one embodiment, the invention provides a cell culture systemcomprising:

-   -   i) an epithelial stem cell or epithelial progenitor cell or a        population of epithelial stem cells or epithelial progenitor        cells;    -   ii) R-spondin 1;    -   iii) CHIR99021;    -   iv) a histone deacetylase inhibitor; and    -   v) optionally an inhibitor of a Bone Morphogenic Protein.

In another embodiment, the invention provides a cell culture systemcomprising:

-   -   i) an epithelial stem cell or epithelial progenitor cell or a        population of epithelial stem cells or epithelial progenitor        cells;    -   ii) R-spondin 1;    -   iii) CHIR99021;    -   iv) an Atoh1 inhibitor; and    -   v) optionally an inhibitor of a Bone Morphogenic Protein.

In yet another embodiment, the invention provides a cell culture systemcomprising:

-   -   i) an epithelial stem cell or epithelial progenitor cell or a        population of epithelial stem cells or epithelial progenitor        cells;    -   ii) R-spondin 1;    -   iii) lithium chloride;    -   iv) a histone deacetylase inhibitor; and    -   v) optionally an inhibitor of a Bone Morphogenic Protein.

In yet another embodiment, the invention provides a cell culture systemcomprising:

-   -   i) an epithelial stem cell or epithelial progenitor cell or a        population of epithelial stem cells or epithelial progenitor        cells;    -   ii) R-spondin 1;    -   iii) a Wnt Agonist;    -   iv) a HDAC6 inhibitor; and    -   v) optionally an inhibitor of a Bone Morphogenic Protein.

In yet another embodiment, cell culture systems of the inventioncomprise epithelial stem cells and populations of epithelial stem cellsthat can comprise LGR5 positive stem cells.

In yet another embodiment, the population of epithelial stem cells orepithelial progenitor cells in the cell culture system the inventioncomprises at least 30%, 85%, 90%, 95% or 99% of the cells in the system.

In yet another embodiment, the invention provides a cell culture systemcomprising:

-   -   i) a tumor organoid;    -   ii) an agent that binds to the Leucine-rich repeat-containing        G-protein coupled receptor 5;    -   iii) a Wnt Agonist;    -   iv) a histone deacetylase inhibitor or an Atoh1 inhibitor; and    -   v) optionally an inhibitor of a Bone Morphogenic Protein.

In yet another embodiment, the invention provides a cell culture systemcomprising a submucosa base, a coating layer comprising collagen and acell layer comprising any member of the group consisting of epithelialstem cells, an isolated tissue comprising epithelial stem cells, and/orepithelial organoids. The coating comprising collagen can be on top of,or surrounding, the epithelial stem cells, isolated tissue comprisingepithelial stem cells, or epithelial organoids. The coating comprisingcollagen can also be between the SIS base and the epithelial stem cells,isolated tissue comprising epithelial stem cells, or epithelialorganoids. The submucosa base can comprise SIS and can further comprisean Epidermal Growth Factor, a Bone Morphogenic Protein, an agent thatbinds to the Leucine-rich repeat-containing G-protein coupled receptor5, a Wnt Agonist, Y-27632 and a histone deacetylase inhibitor. This cellculture system can further comprise cell culture solutions of theinvention, including a solution comprising an inhibitor of a BoneMorphogenic Protein, an agent that binds to the Leucine-richrepeat-containing G-protein coupled receptor 5, a Wnt Agonist, Y-2763and a histone deacetylase inhibitor.

In yet another embodiment, the invention provides a cell culture systemcomprising a submucosa base and epithelial stem cells, isolated tissuecomprising epithelial stem cells, or epithelial organoids, wherein thesubmucosa base comprises an Epidermal Growth Factor, a Bone MorphogenicProtein, R-spondin 1, CHIR99021, Y-27632 and a histone deacetylaseinhibitor. This cell culture system can further comprise a solutioncomprising an Epidermal Growth Factor, an inhibitor of a BoneMorphogenic Protein, R-spondin 1, CHIR99021, Y-27632 and a histonedeacetylase inhibitor.

In yet another aspect, the invention provides methods of formingepithelial organoids from isolated epithelial stem cells.

In one embodiment, the invention provides a method of forming epithelialorganoids from isolated epithelial stem cells with high efficiency, saidmethod comprising the steps of:

-   -   i) incubating isolated epithelial stem cells in the presence of        Noggin, R-spondin 1, CHIR99021 and a histone deacetylase        inhibitor; and    -   ii) forming epithelial organoids from said isolated epithelial        stem cells, wherein at least about 25%, 40%, 50%, 75%, 90%, of        the isolated epithelial stem cells form epithelial organoids.

In another embodiment, the invention provides a method of formingepithelial organoids from a single isolated epithelial stem cell withhigh efficiency, said method comprising the steps of:

-   -   i) incubating said single isolated epithelial stem cell in the        presence of Noggin, R-spondin 1, CHIR99021 and a histone        deacetylase inhibitor; and    -   ii) forming epithelial organoids from said isolated epithelial        stem cell, wherein at least about 6% of the single isolated        epithelial stem cells form epithelial organoids.

In yet another aspect, the invention provides a method of determiningthe efficacy of a chemotherapeutic agent relative to a tumor organoid,said method comprising the steps of:

-   -   i) incubating a tumor organoid in the presence of an inhibitor        of a Bone Morphogenic Protein, R-spondin 1, a Wnt agonist, a        histone deacetylase inhibitor and a chemotherapeutic agent; and    -   ii) measuring a parameter selected from the group consisting of        inhibition of cell viability, inhibition of cell proliferation,        inhibition of tumor associated gene expression, activation of        apoptosis and inhibition of cell survival,

-   wherein detecting an increase in the parameter indicates efficacy of    the chemotherapeutic agent relative to a tumor organoid.

In yet another aspect, the invention provides a method of forming apaneth cell in a cell culture system comprising incubating an epithelialstem cell in the presence of at least one Wnt agonist and at least oneinhibitor of Notch, each in an amount sufficient to produce a panethcell.

In one embodiment, the epithelial stem cell can be further be incubatedin the presence of at least one inhibitor of a Bone Morphogenic Protein.

In another embodiment, the inhibitor of Notch is DAPT.

In yet another embodiment, the epithelial stem cell is an LGR5 positivestem cell.

In yet another aspect, the invention provides a method of forming anenterocyte in a cell culture system comprising incubating an epithelialstem cell in the presence of at least one Wnt inhibitor and at least onehistone deacetylase inhibitor, each in an amount sufficient to producean enterocyte in a cell culture system. The epithelial stem cell can befurther incubated in the presence of an Epidermal Growth Factor and/oran inhibitor of a Bone Morphogenic Protein.

In one embodiment, the Wnt inhibitor can be selected from the groupconsisting of IWP-2, XAV-939, ICG-001, LGK-974, IWR-1-endo, KY02111,Wnt-059, DKK-1, FH-535, Box5, Peptide Pen-N3, Anti-SFRP antibody, andAnti-LRP6 antibody.

In yet another aspect, the invention provides a method of forming agoblet cell in a cell culture system comprising incubating an epithelialstem cell in the presence of at least one Wnt inhibitor and at least oneNotch inhibitor, each in an amount sufficient to produce a goblet cellin a cell culture system. The epithelial stem cell can be furtherincubated in the presence of an Epidermal Growth Factor.

In one embodiment, the Notch inhibitor can be selected from the groupconsisting of DAPT, RO4929097, LY450139, LY900009, LY3039478, LY411575,YO-01027, BMS-708163, BMS-906024, Compound E, BMS-299897, SAHM1,Abeta42-Selective and SB 225002.

In yet another aspect, the invention provides a method of forming anenteroendocrine cell in a culture system comprising incubating anepithelial stem cell in the presence of at least one inhibitor of Notchand an agent that inhibits at least one of a Receptor Tyrosine Kinase, aMitogen-activated protein (MAP) kinase or an Extracellularsignal-regulated kinase (ERK), each in an amount sufficient to producean enteroendocrine cell in a cell culture system. The epithelial stemcell can be further incubated in the presence of an Epidermal GrowthFactor, an agent that binds to the Leucine-rich repeat-containingG-protein coupled receptor 5 and/or an inhibitor of a Bone MorphogenicProtein. The MAP kinase can be Mitogen-activated protein kinase kinase(MEK).

In one embodiment, the agent that inhibits the MAP kinase can beselected from the group consisting of AS-703026, PD0325901, PD98059,Selumetinib, SL-327, U0126, TAK-733 and Trametinib.

In another embodiment, the agent that inhibits an RTK can be selectedfrom the group consisting of Gefitinib, AG 99, Erlotinib, Afatinib,Lapatinib, WZ4002 and AG-18.

In yet another embodiment, the agent that inhibits an ERK can beAS-703026 or PD0325901.

In yet another aspect, the invention provides a method of formingintestinal epithelial cells in a subject in need thereof, comprisingadministering to the subject a Wnt agonist and a histone deacetylaseinhibitor in an amount sufficient to form intestinal epithelial cells inthe subject. The Wnt agonist can be CHIR99021 and the histonedeacetylase inhibitor can be Valproic acid. The CHIR99021 can beadministered in an amount of about 0.1 mg/kg/day to about 100 mg/kg/dayand the Valproic acid can be administered in an amount of about 1mg/kg/day to about 1000 mg/kg/day.

In yet another aspect, the invention provides a method of formingintestinal epithelial cells in a subject in need thereof, comprisingadministering to the subject a Wnt agonist and a Notch agonist in anamount sufficient to form intestinal epithelial cells in the subject.

In yet another aspect, the invention provides a method of treating anintestinal disorder, the method comprising administering to the subjecta Wnt agonist and a histone deacetylase inhibitor or a Wnt agonist and aNotch agonist. In some embodiments, the intestinal disorder is selectedfrom the group consisting of: enterocolitis; viral infections, such asnon-specific enteritis or specific viral enteritis; diverticulitis;bacterial enterocolitis, such as salmonellosis, shigellosis,campylobacter enterocolitis, or yersinia enterocolitis; protozoaninfections such as amebiasis; helminthic infection; andpseudomembraneous colitis and pulmonary complications in cystic fibrosisand chronic obstructive pulmonary disease; appendicitis; atrophicgastritis; Barrett's esophagus; pneumonitis; cervicitis; chronicinterstitial nephritis; colitis; colonic diverticulitis; conjunctivitis;contact dermatitis; Curling's ulcers; Cushing's ulcers; cystitis;gangrene; gingivitis; mastitis; esophagitis; pancreatitis; panniculitis;phlegmonous gastritis; glomerulonephritis; and autoimmune diseasesincluding, but not limited to, inflammatory bowel disease, ulcerativecolitis, Crohn's disease, Addison's disease and glomerulonephritis(e.g., crescentic glomerulonephritis, proliferative glomerulonephritis).

Other features and advantages of the invention will be apparent from thefollowing detailed description and figures, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The following Detailed Description, given by way of example, but notintended to limit the invention to specific embodiments described, maybe understood in conjunction with the accompanying figures, incorporatedherein by reference.

FIG. 1 depicts scattered expression of Lgr5-GFP in vivo. Small intestinewas harvested from Lgr5-GFP mice and directly imaged under fluorescencemicroscopy. While all areas of the small intestine were covered bycrypts, approximately half of these crypts contained GFP+ cells. Scalebar: 100 μm.

FIGS. 2A-2H show the combination of CHIR and VPA promoting theproliferation and self-renewal of Lgr5+ Stem Cells. FIG. 2A depicts GFPand bright field images of small intestinal crypts cultured for 6 daysin the presence of ENR (EGF, Noggin and R-spondin1), ENR+VPA (ENR-V),ENR+CHIR (ENR-C) and ENR+VPA+CHIR (ENR-CV). Apoptotic cells are visiblein the lumen with autofluorescence (red arrows), and white arrowsindicate specific Lgr5-GFP at the base of crypts. Scale Bar: 100 μm.FIG. 2B depicts quantification of cell proliferation and GFP expressionof crypts cultured in multiple conditions. Crypts were cultured in 24well plates for 6 days and dissociated into single cells using Accutase.Live cell numbers in each well were counted as an indicator of cellproliferation. Lgr5-GFP expression was measured by flow cytometryanalysis. Error bars indicate S.D. of triplicate wells. Experiments wereperformed 3 times and showed similar results. FIGS. 2C and 2D depictflow cytometry analysis of GFP expression of single Lgr5-GFP cells after7 days of culture under multiple conditions as indicated. Error barsindicate S.D. of triplicate wells. FIG. 2E depicts GFP and brightfieldimages of single Lgr5-GFP cells following 9 days of culture. Scale Bar:100 μm. FIG. 2F depicts representative images of 4,000 FACS isolatedsingle Lgr5-GFP cells cultured under multiple conditions for 7 days andFIG. 2G depicts quantification of colony numbers. Error bars indicateS.D. of triplicate wells. FIG. 2H depicts the metaphase spread of a cellcultured in the CV condition for 80 days having a normal karyotype(2n=40). (Unless otherwise indicated, in all panels: ***P<0.001;**P<0.01; *P<0.05; NS P>0.05.)

FIGS. 3A-3G depict cell growth and GFP expression as a function ofculture conditions.

-   FIGS. 3A and 3B depict colony numbers and live single cell numbers,    respectively, from triplicate wells enumerated at each time point.    Error bars indicate S.D. In FIG. 3A, the series are, from left to    right, Day 0, Day 2, Day 4, Day 6, Day 8, and Day 10. FIG. 3C    depicts FACS sorting of freshly isolated single Lgr5-GFP+ cells. A    GFPhigh single cell population was collected. Representative FACS    analysis and the gating strategy to define the GFP+ cell population    is shown. Freshly isolated single cells from crypts showed two    discriminated GFP^(high) and GFP^(low) populations while cultured    cells did not show discriminated GFP^(high) and GFP^(low)    populations, so all GFP+ cells were gated for analysis. Note that    the ENR-CV cultured cells showed a single GFP+ population that was    GFP highly positive. The GFP-population represents Lgr5− cells as    well as Lgr5+/GFP− cells (i.e. GFP silenced stem cells), which is    present in all unsorted crypt cultures, but not in sorted single    Lgr5-GFP cell cultures (See FIG. 2C). A total of 10,000 live cells    were analyzed for each sample. FIG. 3D depicts growth factor    requirements for self-renewal of Lgr5+ stem cells in the CV culture    condition. Crypts were cultured for 6 days in the presence of CHIR    and VPA, with EGF, Noggin, R-spondin 1 and their combinations, as    indicated. E: EGF (50 ng/ml); N: Noggin (100 ng/ml); R: R-spondin 1    (500 ng/ml); C: CHIR (3 μM); V: VPA (1 mM). FIG. 3E depicts crypts    cultured for 6 days in multiple conditions as indicated. GFP and    brightfield images are shown. Scale bars: 200 μm. FIG. 3F depicts    morphology and Lgr5-GFP expression of colon crypts cultured in ENR,    ENR-C and ENR-CV conditions. FIG. 3G depicts isolated number of    organoids formed at day 5 in the presence of EGF, Noggin and    R-spondin 1 or R-spondin 2 at multiple concentrations. All scale    bars: 200 μm.

FIGS. 4A-4B depict testing of multiple culture conditions for EPHB2+human colonic stem cells. Crypts were cultured for 6 days in multipleconditions as indicated. GFP and brightfield images are shown in FIG.4A. W: Wnt3a (100 ng/ml); Ni: Nicotinamide (10 mM); P: PGE2 (0.02 μM);A: A-83-01 (0.5 μM); S: SB202190 (10 μM); V: Valproic Acid or VPA (1mM). The EGF, Noggin, R-spondin 1, Wnt3a and VPA, or ENR-W-V, conditionserves as a control to show increased expression of GFP. Scale bars: 200μm. FIG. 4B depicts quantification of cell proliferation and GFPexpression of crypts cultured under multiple conditions. Crypts werecultured for 6 days in 24-well plates and dissociated into single cells.Live cell numbers in each well were counted and the percentage of GFP+cells was analyzed by flow cytometry. Error bars indicate S.D. oftriplicate wells.

FIGS. 5A-5D depicts culture of single Lgr5-GFP stem cells. FIG. 5Adepicts single isolated Lgr5-GFP+ cells cultured for 9 days in CVcondition. Scale bars: 200 μm. FIG. 5B depicts 1500 FACS sorted singleLgr5+ cells cultured in Matrigel under conditions as indicated.Representative images from day 7 cultures are shown. FIG. 5C indicatesquantification of colony numbers. Error bars indicate S.D. or triplicatewells. FIG. 5D shows sorted single Lgr5+ stem cells seeded in 48-wellplates. Viable cell numbers were quantified at 12 hours after plating.Colony numbers were counted at day 7 and colony-forming efficiency wasquantified. V: VPA; C: CHIR; W: Wnt3a at 100 ng/ml. Error bars indicateS.D. of 3 triplicate wells. Experiments were performed 3× and showedsimilar results.

FIGS. 6A-6D depict maintenance of Lgr5+ stem cell self-renewal by thecombination of CHIR and VPA. Confocal images of Lysozyme (FIG. 6A), Ki67(FIG. 6B) and EdU (FIG. 6C) staining of organoids cultured under the ENRcondition (Upper panels) and colonies cultured in the ENR-CV condition(Lower panels) are shown. For EdU staining, cells were cultured with thethymidine analogue EdU (red) for 1 hour. In FIGS. 6B and 6C, only cryptdomains contain Ki67 positive cells or incorporate EdU in the ENRcondition (Upper panels) while Ki67 or EdU is present throughout thecell aggregates in the CV condition (Lower panels). FIG. 6D depictsquantitative real-time PCR analysis of relative mRNA expression ofmarkers for mature intestinal epithelial cells cultured for 6 days underconditions as indicated (Intestinal Alkaline phosphatase [Alpi] forenterocytes, Mucin 2 [Muc2] for goblet cells, Chromogranin A [ChgA] forenteroendocrine cells, Lysozyme [Lyz] for Paneth cells, and Lgr5 forintestinal stem cells). ENR-CV(D40) indicates cells that were culturedin the CV condition for 40 days. Scale bars: 50 μm. In FIG. 6D, theseries are, from left to right, Alpi, Muc2, ChgA, Lyz, and Lgr5.

FIGS. 7A-7D depict differentiation of intestinal stem cells culturedunder CV Condition. FIG. 7A depicts staining of differentiation markers(Alp for enterocytes, Muc2 for goblet cells (white arrows) and mucinsecreted by goblet cells, ChgA for enteroendocrine cells and Lyz forPaneth cells) of cells transferred from CV condition to ENR conditionand cultured for 4 days. DAPI was used to stain nuclei and GFP indicatesthe presence of stem cells. FIG. 7B shows real-time RT-PCR analysis ofrelative mRNA expression of mature intestinal epithelial markers fromcells cultured under multiple conditions. Cells were initially culturedfrom single cells in the CV condition for 6 days. Cell colonies werethen harvested, washed and re-plated into several wells of 24 wellplates and cultured for 4 days in Matrigel under multiple conditions asindicated. ENR was added in all conditions and the cells cultured withENR alone were used as controls. I: IWP-2 (204), D: DAPT (10 μM), C:CHIR (3 μM), V: VPA (1 mM). Error bars indicate S.D. FIG. 7C depicts Alpstaining of cells cultured under multiple conditions. There is a clearmorphology change of cells in the ID and CD conditions, which resemblesgoblet cells and Paneth cells. Scale bars: 50 μm. FIG. 7D depictsimmunocytochemistry staining of differentiation markers. Cells culturedunder CD and ID conditions were used for Mucin 2 (Muc2), Chromogranin A(ChgA) and Lysozyme (Lyz) staining. Three dimensional reconstructedconfocal images are shown. Scale bars: 50 μm.

FIGS. 8A-8F depict controlled differentiation of Lgr5+ stem cells invitro. FIG. 8A depicts staining of organoids cultured in ENR condition.The left panel depicts Alp staining of enterocytes. Prior to staining,the organoid was cut open under a dissecting microscope by using a sharpblade and the luminal content was removed. The middle panel depicts Muc2staining of goblet cells (arrows) as well as mucin secreted by gobletcells, and the right panel depicts ChgA staining of enteroendocrinecells. GFP+ cells indicate Lgr5+ stem cells. FIG. 8B provides a schemeof the differentiation protocol. Single Lgr5+ stem cells were culturedin the CV condition for 4-6 days to form colonies. Cell colonies werethen harvested, washed, embedded within fresh Matrigel and culturedunder multiple conditions. FIG. 8C depicts morphology of cell coloniestransferred from the CV condition to the ENR condition and cultured for4 days (upper panels). Colonies continuously cultured in the CVcondition are shown as a control (lower panels). FIG. 8D depictsmorphology of differentiated cells with low and high magnificationimages for each condition. Note the clear change in morphology for mostcells in the CD and ID conditions, which reflects formation of Panethcells and goblet cells, respectively. FIG. 8E depicts Alp staining ofcolonies cultured in IV condition. Apical (left panel) and homogeneous(right panel) staining of Alp are shown. FIG. 8F depicts Muc2 stainingof colonies cultured in ID and CD conditions. All scale bars: 50 μm.

FIGS. 9A-9F depicts a Mechanism of Action for CHIR and VPA. FIG. 9Adepicts morphology and Lgr5-GFP expression of crypts cultured inmultiple conditions for 6 days. C: CHIR (3 μM); Li: LiCl (5 mM); W:Wnt3a (100 nM). FIG. 9B depicts cell numbers and percentage of GFP+cells for 6 day crypt cultures. The data is representative of threeindependent experiments. FIG. 9C shows 6 day cultures of crypts in ENR-C(Control) condition or together with HDAC inhibitors. FIG. 9D depictsquantification of GFP percentage, total live cell number and relativeGFP intensity of cells in FIG. 9C. FIG. 9E depicts the effects of VPAand TSA on cell proliferation and GFP expression at multipleconcentrations. FIG. 9F depicts the effects of nicotinamide (Ni) incombination with Wnt3a (W, 100 ng/ml) or CHIR (C, 3 Shown are cellnumbers and percentage of GFP+ cells of crypts cultured for 6 days inmultiple conditions. (Unless otherwise indicated, in all panels: Errorbars indicate S.D. or triplicate wells. ***P<0.001; **P<0.01; *P<0.05;NS P>0.05.)

FIG. 10 depicts morphology and GFP expression of single Lgr5-GFP cellscultured in multiple conditions. Scale bars: 100 μm.

FIGS. 11A-11D depict mechanism of VPA. FIG. 11A depicts VPA rescuing GFPexpression following Notch inhibition. Crypts were cultured in ENR-Ccondition with or without DAPT (D, 5 μM) and varying concentration ofVPA (V, 0.25-4 mM) for 3 days. Scale bars: 200 μm. FIG. 11B and 11Cdepict crypts cultured in the ENR (FIG. 11B) or ENR+CHIR (FIG. 11C)conditions for 4 days followed by addition of VPA at differentconcentrations for another 24 hours. The expression of Notch1, Hes1 andAtoh1 were analyzed by Real-time RT-PCR. FIG. 11D depicts analysis ofNotch1, Hes1 and Atoh1 expression by Real-time RT-PCR in crypts after 6days of culture. In FIG. 11B-11C, the series are, from left to right, 0,0.5, 1, 2, and 3. In FIG. 11D, the series are, from left to right, ENR,ENR-V, ENR-C, and ENR-CV.

FIGS. 12A-12B depict a model for Intestinal Stem Cell self-renewal anddifferentiation (FIG. 12A) under physiological conditions and (FIG. 12B)in vitro culture.

FIGS. 13A-13B depict the combination of CHIR and VPA promoting theproliferation and GFP expression of Lgr5+ stem/progenitor cells derivedfrom the mouse inner ear. FIG. 13A depicts brightfield and GFP images ofisolated cochlea sensory epithelium derived from an Lgr5-GFP mouse atpost natal day 2. FIG. 13B depicts isolated cochlea sensory epitheliumdissociated into single cells and cultured in multiple conditions for 11days. E: EGF; N: Noggin; R: R-spondin 1; C: CHIR99021, V: VPA. Scalebars: 100 μm.

FIGS. 14A-14F depict the combination of CHIR and VPA promoting theproliferation and GFP expression of Lgr5+ stem/progenitor cells from themouse inner ear. FIG. 14A depicts GFP expression of inner ear epithelialcells. FIG. 14B depicts quantification of GFP expression and cellnumber. FIG. 14C depicts brightfield and GFP images. FIG. 14D depictscell number of 8 day cultures of inner ear stem cells in multipleconditions as indicated. FIG. 14E depicts GFP percentage of 8 daycultures of inner ear stem cells in multiple conditions as indicated.FIG. 14F depicts morphology and GFP expression of Lgr5-GFP inner earstem cells cultured in multiple conditions. All scale bars: 200 μm.

FIG. 15 depicts seeding of murine small intestinal crypts on healthymouse colon tissue in vitro. The left panel shows isolated smallintestinal crypts placed onto colon with partially denuded epithelium.White arrows indicate the seeded crypts. The right panel shows seededcrypts attached to the colon and spreading across its surface after 24hours. Black arrows indicate the same location as white arrows in leftpanel.

FIG. 16 depicts engraftment of crypts 48 hours after seeding.Fluorescent (top panel) and brightfield (bottom panel) images of mousecolon tissue seeded with crypts are shown. Crypts were stained with DiDprior to seeding. White lines indicate areas that include engraftedcells.

FIG. 17 depicts engraftment of crypts following 6 days of in vitroculture. Shown are GFP (left panel), RFP (middle panel) and brightfield(right panel) channel images of mouse colon tissue seeded with crypts.GFP signal indicates the presence of Lgr5cells.

FIG. 18 depicts a confocal image of excised prolapsed ulcerative colitistissue from the TRUC mouse with engrafted crypts. Crypts were stainedwith DiD prior to seeding. Prolapsed tissue is shown via greenautofluorescence.

FIGS. 19A-19N depict a schematic of seeding (left) and post-incubationorganoid growth (right) in culture systems evaluated. FIGS. 19A and 19Bdepict a typical submucosal seeding method (herein referred to as “barepatch”), which supports monolayer growth and organoid dissociation.FIGS. 19C and 19D depict GF-infused SIS (GFs include EGF, Noggin,R-spondin 1, Y-27632, Valproic Acid, CHIR) to support 3-dimensionalorganoid growth. FIGS. 19E and 19F depict gel-patch composed ofGF-infused SIS with a collagen overlay. FIG. 19E, inset, depicts eachorganoid individually encased in a soft gel as well as SIS base layer.FIGS. 19G and 19H depict typical collagen suspension with GFs (EGF,Noggin, R-spondin 1, Y-27632, Valproic Acid, CHIR) added directly toculture media. FIGS. 19I and 19J depict typical collagen suspension withGFs (EGF, Noggin, R-spondin 1, Y-27632, Valproic Acid, CHIR) embedded inthe gel. FIGS. 19K and 19L depict typical collagen suspension withoutadditional GFs added to culture media. FIGS. 19M and 19N depict typicalMatrigel suspension without additional GFs added to media (experimentalcontrol).

FIG. 20A depicts a schematic of the seeding procedure with Lgr5+organoids; patterned circles represent infused growth factors (EGF,Noggin, R-spondin 1, Y-27632, Valproic Acid and CHIR). FIG. 20B depictsan initial adherence phase with arrows depicting growth embedded factordiffusion support. FIG. 20C depicts complete culture system withcollagen overlay, thickness measurements depicted.

FIG. 21A provides a comparison of organoid growth in 7 culture systems.The series are, from left to right, Matrigel, Gel-Patch with GFs, BarePatch with GFs, Collagen I, Collagen I with Media GFs, Collagen I withembedded GFs, and Bare patch without GFs. FIG. 21B and FIG. 21C show arepresentative organoid at 48 hours from the gel-patch system with GFs,with GFP+ fluorescence indicating Lgr5+ stem cells present in the cryptbases (some central autofluorescence is seen). *=p<0.05 at 24 hours(collagen I (CI) vs. all), 48 hours (bare patch with GFs (BPGF) vs. all;CI vs. Matrigel (M); CI with GFs (CIGF) vs. M, CI, Collagen I withembedded GFs (CIEGF), bare patch (BP), gel-patch system with GFs(PSGF)), 72 hours (CI vs. all; BP, CIEGF, and CIGF vs. M, PS, BPGF, CI)and 96 hours (CI vs. all, BP, CIEGF, and CIGF vs. all). Scale bar (FIG.21B and 21C)=200 um.

FIG. 22 depicts successful growth and crypt expansion of a seededorganoid in the Gel-patch with GFs system. A representative sequence ofimages depicting ex-vivo expansion of a seeded organoid on the SIS patchsystem with GFs is shown. Out-of-focus crypts are an effect of growth in3-dimensions viewed in single plane microscopy.

FIG. 23A provides a schematic showing the creation of a 4mm gastricdefect. Placement of a 6 mm patch over the defect is shown in FIG. 23B.As shown in FIG. 23C, there was no visible defect on the externalgastric wall, as indicated on a representative stomach sample at 1 weekpost-op. Gross defects (arrows), as viewed from the internal gastricwall, are displayed according to the type of patch placed: FIG. 23Dshows SIS patch without GFs, FIG. 23E shows SIS patch plus GFs and FIG.23F shows PGSU backing only, without SIS. The SIS patch with GFs showedcomplete closure and epithelialization of the gastric wall effect,whereas defects remained partially open in SIS only and completely openin PGSU without SIS.

FIG. 24 depicts real-time RT-PCR analysis of marker gene expression ofisolated human intestinal crypts cultured in multiple conditions. EGF,Noggin and R-spondin1 were added to all conditions. C: CHIR, Ni:Nicotinamide, W: Wnt3a, A: A83-01, S: SB202190, P: PGE2, V: VPA, Tu:Tubastatin A, Crypt indicates freshly isolated human small intestinalcrypts. Error bars indicate S.D., n=3.

FIGS. 25A-25B depict optimizing culture condition for human intestinalstem cells. FIG. 25A depicts proliferation of human intestinalepithelial cells cultured in multiple conditions. Freshly isolated humansmall intestinal crypts were cultured in multiple conditions asindicated. EGF, Noggin, R-spondin1 were present in all conditions. Cellnumbers were quantified at day 9 after seeding. C: CHIR, V: VPA, used at0.5-1.5 mM, Ni: Nicotinamide. FIG. 25B depicts LGR5 expression of cellscultured in multiple conditions as in FIG. 15A. VPA was used at 1 mM.

FIG. 26 depicts human intestinal stem cell culture. Cells were culturedin human intestinal stem cell culture media (containing EGF, Noggin,R-Spondin1, CHIR99021, VPA and Nicotinamide). Shown are cells of passage2 at day 5 after passage. Scale bars: 400 μm.

FIG. 27 depicts increased crypt size following administration of CHIRand VPA over the course of 7 days in vivo in an animal model system.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein, an “antibody” is any immunoglobulin polypeptide, orfragment thereof, having immunogen binding ability.

As used herein, an “agonist” is an agent that causes an increase in theexpression or activity of a target gene or protein, respectively. Anagonist can bind to and activate its cognate receptor in some fashion,which directly or indirectly brings about this physiological effect onthe target gene or protein.

As used herein, an “inhibitor” is an agent that causes a decrease in theexpression or activity of a target gene or protein, respectively. An“antagonist” can be an inhibitor, but is more specifically an agent thatbinds to a receptor, and which in turn decreases or eliminates bindingby other molecules.

As used herein, an “inhibitory nucleic acid” is a double-stranded RNA,siRNA, shRNA, or antisense RNA, or a portion thereof, or a mimeticthereof, that when administered to a mammalian cell results in adecrease in the expression of a target gene. Typically, a nucleic acidinhibitor comprises at least a portion of a target nucleic acidmolecule, or an ortholog thereof, or comprises at least a portion of thecomplementary strand of a target nucleic acid molecule. Typically,expression of a target gene is reduced by 10%, 25%, 50%, 75%, or even90-100%.

By “anti-sense” is meant a nucleic acid sequence, regardless of length,that is complementary to the coding strand or mRNA of a nucleic acidsequence. As referred to herein, a “complementary nucleic acid sequence”is a nucleic acid sequence capable of hybridizing with another nucleicacid sequence comprised of complementary nucleotide base pairs. By“hybridize” is meant pair to form a double-stranded molecule betweencomplementary nucleotide bases (e.g., adenine (A) forms a base pair withthymine (T), as does guanine (G) with cytosine (C) in DNA) undersuitable conditions of stringency. (See, e.g., Wahl, G. M. and S. L.Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) MethodsEnzymol. 152:507). In one embodiment, an antisense RNA is introduced toan individual cell, tissue or organanoid. The anti-sense nucleic acidmay contain a modified backbone, for example, phosphorothioate,phosphorodithioate, or other modified backbones known in the art, or maycontain non-natural internucleoside linkages.

By “siRNA” is meant a double stranded RNA. Optimally, an siRNA is 18,19, 20, 21, 22, 23 or 24 nucleotides in length and has a 2 base overhangat its 3′ end. These dsRNAs can be introduced to an individual cell orculture system. Such siRNAs are used to downregulate mRNA levels orpromoter activity.

As used herein, a “fragment” is a portion of a polypeptide or nucleicacid molecule. This portion contains, preferably, at least 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of thereference nucleic acid molecule or polypeptide. A fragment may contain10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600,700, 800, 900, or 1000 nucleotides or amino acids

As used herein, the term “stem cell” refers to a multipotent cell havingthe capacity to self-renew and to differentiate into multiple celllineages.

As used herein, the term “epithelial stem cell” refers to a multipotentcell which has the potential to become committed to multiple celllineages, including cell lineages resulting in epithelial cells.

As used herein, the term “progenitor cell” refers to alineage-restricted cell derived from a stem cell.

As used herein, the term “epithelial progenitor cell” refers to amultipotent cell which has the potential to become restricted to celllineages resulting in epithelial cells.

As used herein, the term “self-renewal” refers to the process by which astem cell divides to generate one (asymmetric division) or two(symmetric division) daughter cells with development potentials that areindistinguishable from those of the mother cell. Self-renewal involvesboth proliferation and the maintenance of an undifferentiated state.

As used herein, the term “engraft” or “engraftment” refers to theprocess of stem or progenitor cell incorporation into a tissue ofinterest in vivo through contact with existing cells of the tissue.

As used herein, the term “isolated” refers to a material that is free tovarying degrees from components which normally accompany it as found inits native state. “Isolate” denotes a degree of separation from originalsource or surroundings.

As used herein, a “population” of cells is any number of cells greaterthan 1, but is preferably at least 1X10³ cells, at least 1X10⁴ cells, atleast at least 1X10⁵ cells, at least 1X10⁶ cells, at least 1X10⁷ cells,at least 1X10⁸ cells, at least 1X10⁹ cells, or at least 1X10¹⁰ cells.

As used herein, the term “organoid” or “epithelial organoid” refers to acell cluster or aggregate that resembles an organ, or part of an organ,and possesses cell types relevant to that particular organ.

As used herein, a “subject” is a vertebrate, including any member of theclass mammalia.

As used herein, a “mammal” refers to any mammal including but notlimited to human, mouse, rat, sheep, monkey, goat, rabbit, hamster,horse, cow or pig.

A “non-human mammal”, as used herein, refers to any mammal that is not ahuman.

As used herein, “increasing” refers to increasing by at least 5%, forexample, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 95, 99, 100% or more, for example, as compared tothe level of a reference.

As used herein, “increases” also means increases by at least 1-fold, forexample, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80,90, 100, 200, 500, 1000-fold or more, for example, as compared to thelevel of a as compared to the level of a reference standard.

As used herein, “decreasing” refers to decreasing by at least 5%, forexample, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 95, 99 or 100%, for example, as compared to thelevel of reference.

As used herein, “decreases” also means decreases by at least 1-fold, forexample, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80,90, 100, 200, 500, 1000-fold or more, for example, as compared to thelevel of a reference.

As used herein, the term “reference” means a standard or controlcondition (e.g., untreated with a test agent or combination of testagents).

As used herein, the term “eliminate” means to decrease to a level thatis undetectable.

As used herein, the term “synergy” or “synergistic effect” is an effectwhich is greater than the sum of each of the effects taken separately; agreater than additive effect.

As used herein, the terms “treat,” “treating,” “treatment,” and the likerefer to reducing or ameliorating a disorder and/or symptoms associatedtherewith. It will be appreciated that, although not precluded, treatinga disorder or condition does not require that the disorder, condition orsymptoms associated therewith be completely eliminated.

In this disclosure, “comprises,” “comprising,” “containing” and “having”and the like can have the meaning ascribed to them in U.S. Patent lawand can mean “includes,” “including,” and the like; “consistingessentially of” or “consists essentially” likewise has the meaningascribed in U.S. Patent law and the term is open-ended, allowing for thepresence of more than that which is recited so long as basic or novelcharacteristics of that which is recited is not changed by the presenceof more than that which is recited, but excludes prior art embodiments.

Other definitions appear in context throughout this disclosure. Unlessotherwise defined, all technical and scientific terms used herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this invention belongs. In case of conflict, the presentspecification, including definitions, will control.

Methods and Compositions of the Invention

I. Cell Culture Solutions and Systems

Cell culture solutions and systems to promote homogeneous epithelialstem cell cultures, efficient epithelial organoid formation and scale-upof the same for use in transplantation have now been discovered.

Cell culture solutions comprising an inhibitor of a Bone MorphogenicProtein, an inhibitor of Glycogen synthase kinase-3 beta (GSK3 (3), anagent that binds to the Leucine-rich repeat-containing G-protein coupledreceptor 5 (LGR5) and a histone deacetylase inhibitor can be utilized toform epithelial cell colonies from isolated epithelial stem cells. Inspecific embodiments, at least about 25%, about 40%, about 50%, about75%, about 90% to about 100% of isolated epithelial stem cells formepithelial cell colonies in the presence of this cell culture solution.In addition, at least about 6% of single isolated epithelial stem cellsform epithelial cell colonies in the presence of this cell culturesolution. Combinations of 1,6-[[2-[[4-(2,4-Dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile“CHIR99021” (Ring et al., 2003), an inhibitor of Glycogen synthasekinase-3 beta and valproic acid, a histone deacetylase inhibitor, havesynergistic effects on colony forming efficiency.

Bone Morphogenic Proteins (BMPs) are members of the TGF-beta superfamilyand comprise metalloproteases implicated in embryonic patterning amongdiverse species as well as post-embryonic cell signaling. Inhibitors ofBMPs include, for example, agents that bind to a BMP molecule to form acomplex wherein the BMP activity is decreased or eliminated, for exampleby preventing or inhibiting the binding of the BMP molecule to a BMPreceptor. Alternatively, the inhibitor is an agent that acts as anantagonist or reverse agonist. This type of inhibitor binds to a BMPreceptor and prevents binding of a BMP to the receptor. An example of alatter agent is an antibody that binds a BMP receptor and preventsbinding of BMP to the antibody-bound receptor. Inhibitors of BMPs arewell known in the art (Rider et al., 2010) and can include, but are notlimited to, Noggin, Chordin, Follistatin (Schneyer et al., 1994), DAN,proteins comprising a DAN cysteine-knot domain (including Cerberus andGremlin), Sclerostin, Twisted Gastrulation, UterineSensitivity-Associated Gene-1, Connective-Tissue Growth Factor (Abreu etal., 2002), Inhibin (Wiater and Vale, 2003), BMP-3 (Gamer et al., 2005),Dorsomorphin (Yu et al., 2008) and derivatives, including DMH1 (Hao etal., 2010) and LDN-193189 (Cuny et al., 2008).

Glycogen synthase kinase-3 (GSK3) is a proline-directed serine-threoninekinase that was initially identified as a phosphorylating andinactivating glycogen synthase having two known isoforms, alpha (GSK3A)and beta (GSK-3β). Wnt agonists comprising GSK-3β inhibitors are wellknown in the art and include, but are not limited to, 1,6-[[2-[[4-(2,4-Dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile“CHIR99021” (Ring et al., 2003), LiCl (Klein et al., 1996),BIO-acetoxime ((2′Z,3′E)-6-Bromoindirubin-3′-oxime) (Meijer et al.,2003),N6-[2-[[4-(2,4-Dichlorophenyl)-5-(1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]-3-nitro-2,6-pyridinediamine“CHIR98014” (Ring et al., 2003),3-(2,4-Dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione“SB 216763” also known as GSK-3 Inhibitor IV (Coghlan et al., 2000),3-[(3-Chloro-4-hydroxyphenyl)amino]-4-(2-nitrophenyl)-1H-pyrrol-2,5-dione“SB 415286” (Coghlan et al., 2000),5-ethyl-7,8-dimethoxy-1H-pyrrolo[3,4-c]-isoquinoline-1,3-(2H)-dione“3F8” (Zhong et al., 2009),9-Bromo-7,12-dihydro-indolo[3,2-d][1]benzazepin-6(5H)-one “Kenpaullone”(Schultz et al.,1999; Zaharevitz et al., 1999),9-Bromo-7,12-dihydro-pyrido[3′,2′:2,3]azepino[4,5-b]indol-6(5H)-one“1-Azakenpaullone” (Schultz et al., 1999; Zaharevitz et al., 1999),N-(3-Chloro-4-methylphenyl)-5-(4-ni¬trophenyl)-1,3,4-oxadiazol-2-amine“TC-G 24” (Khanfar et al., 2010),2-Methyl-5-[3-[4-(methylsulfinyl)phenyl]-5-benzofuranyl]-1,3,4-oxadiazole“TCS 2002” (Saitoh et al., 2009),N-[(4-Methoxyphenyl)methyl]-N′-(5-nitro-2-thiazolyl)urea “AR-A 014418”(Bhat et al., 2003),3-[5-[4-(2-Hydroxy-2-methyl-1-oxopropyl)-1-piperazinyl]-2-(trifluoromethyl)phenyl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione“TCS 21311” (Thoma et al., 2011),3-[[6-(3-aminophenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl]oxy]-phenol “TWS119” (Ding et al., 2003), ((2′Z,3′E)-6-Bromoindirubin-3′-acetoxime)“BIO-acetoxime” also known as GSK-3 Inhibitor IX (Meijer et al., 2003),4-(2-Amino-4-oxo-2-imidazolin-5-ylidene)-2-bromo-4,5,6,7-tetrahydropyrrolo[2,3-c]azepin-8-one“10Z-Hymenialdisine” (Breton et al., 1997),2-[(3-iodophenyl)methylsulfanyl]-5-pyridin-4-yl-1,3,4-oxadiazole, alsoknown as GSK-3β Inhibitor II (Wada, 2009),4-Benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione, also known as GSK-3βInhibitor I (Wada, 2009),3-Amino-6-(4-((4-methylpiperazin-1-yl)sulfonyl)phenyl)-N-(pyridin-3-yl)pyrazine-2-carboxamide,HCl , also known as GSK-3β Inhibitor XXVII (US Patent Pub. No.2006/0173014), 4,5-bis(1-Methyl-1H-indol-3-yl)-1,2-dihydropyrazol-3-one,also known as GSK-30 Inhibitor XXVI (Chen et al, 2011), FRATtide peptideSQPETRTGDDDPHRLLQQLVLSGNLIKEAVRRLHSRRLQ (SEQ ID NO: 1) (Bax et al.,2001), 3-Amino-1H-pyrazolo[3,4-b]quinoxaline “Cdk1/5 Inhibitor”(Andreani et al., 1996, 2000; Katoh et al., 2011) and4-((5-Bromo-2-pyridinyl)amino)-4-oxobutanoic acid “Bikinin” (De Rybel etal., 2009). Preferably, the inhibitor of GSK-3β is CHIR99021.

Leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5receptor) is known for its restricted crypt expression and marking ofstem cells in multiple adult tissues and cancers. Agents that bind tothe LGR5 receptor include but are not limited to R-spondins (Kim et al.,2006; Nam et al., 2006), such as R-spondin 1, R-spondin 2, R-spondin 3and R-spondin 4. Preferably, the agent that binds to the LGR5 receptoris R-spondin 1.

In alternative embodiments, lithium chloride (LiCl) can be substitutedfor CHIR99021 or at least about 3 uM CHIR99021 can be substituted forR-spondin 1.

Histones are nuclear proteins that bind DNA and form nucleosomes. Theyare directly involved with both the packaging of DNA into chromosomesand the regulation of transcription. Histone acetylation/deacetylationis a major factor in regulating chromatin structural dynamics duringtranscription. Histone deacetylase inhibitors, which decrease oreliminate histone deacetylation, are well known in the art and caninclude, but are not limited to, Pan-HDAC inhibitors, such as Valproicacid, Trichostatin A, suberoylanilide hydroxamic acid andSuberohydroxamic acid (SBHA), and HDAC6 inhibitors, such as Tubacin,Tubastatin A and Compound 7.

In alternative embodiments, an Atoh1 inhibitor can augment, or besubstituted for, a Histone deacetylase inhibitor. Atoh1 inhibitorsinclude, for example, inhibitory nucleic acids that cause a decrease orelimination in the expression of Atoh1. Inhibitory nucleic acids thattarget Atoh1 are known in the art (Shi et al., 2010).

Cell culture solutions can optionally include an Epidermal Growth Factorand/or a Notch agonist. Epidermal Growth Factor is a cell signalingmolecule involved in diverse cellular functions, including cellproliferation, differentiation, motility, and survival, and in tissuedevelopment. Notch proteins are single-pass transmembrane receptors thatregulate cell fate decisions during development. A Notch agonistincludes, for example, an agent that increases Notch activity in a cell.Notch agonists are well known in the art and can include, but are notlimited to, a Notch1 antibody (N1 Ab), Delta 1, Delta-like 3, Delta-like4, Jagged 1, Jagged 2, DSL peptide and Delta D.

In specific embodiments, the cell culture solution comprises betweenabout 5 to about 500 ng/ml EGF, about 5 to about 500 ng/ml Noggin, about50 to about 1000 ng/ml R-spondin, about 0.1 to about 10 μM CHIR99021 andabout 0.1 to about 5 mM Valproic acid.

In other embodiments, the combination of a Wnt agonist and an HDAC6inhibitor in the cell culture solution is preferred. Accordingly, a cellculture solution can comprise an inhibitor of a Bone MorphogenicProtein, R-spondin 1, a Wnt agonist and a HDAC6 inhibitor.

Wnt proteins are extracellular signaling molecules involved in thecontrol of embryonic development. Wnt agonists are well known in the artand include, but are not limited to, Wnt-1/Int-1(Nusse et al., 1982),Wnt-2/Irp (Int-I-related Protein) (Wainwright et al., 1988), Wnt-2b/13(Katoh et al., 1996), Wnt-3/Int-4 (Katoh et al., 2001), Wnt-3a (Saitohet al., 2001), Wnt-4 (Smolich et al., 1993), Wnt-5a (Burrus et al.,1995), Wnt-5b (Burrus et al., 1995), Wnt-6 (Burrus et al., 1995), Wnt-7a(Smolich et al., 1993), Wnt-7b (Burrus et al., 1995), Wnt-8a/8d (Saitohet al., 2001), Wnt-8b (Lako et al., 1998), Wnt-9a/14 (Bergstein et al.,1997), Wnt-9b/14b/15 (Bergstein et al., 1997), Wnt-10a (Wang et al.,1996), Wnt-10b/12 (Wang et al., 1996), Wnt-11 (Lako et al., 1998),Wnt-16 (Bergstein et al., 1997; Fear et al., 2000), R-spondin 1,R-spondin 2, R-spondin 3, R-spondin 4, Norrin (Planutis et al., 2007),CHIR99021, LiCl, BIO ((27,3′E)-6-Bromoindirubin-3′-oxime), CHIR98014, SB216763, SB 415286, 3F8, Kenpaullone, 1-Azakenpaullone, TC-G 24, TCS2002, AR-A 014418,2-amino-4-[3,4-(methylenedioxy)benzyl-amino]-6-(3-methoxyphenyl)pyrimidine(Liu et al., 2005),2-[2-(4-Acetylphenyl)diazenyl]-2-(3,4-dihydro-3,3-dimethyl-1(2H)-isoquinolinylidene)acetamide“IQ 1” (Miyabayashi et al., 2007),(3α,5β,12α,20R)-3,12-dihydroxycholan-24-oic acid “DCA” (Pai et al.,2004),(2S)-2-[2-(Indan-5-yloxy)-9-(1,1′-biphenyl-4-yl)methyl)-9H-purin-6-ylamino]-3-phenyl-propan-1-ol“QS 11” (Zhang et al., 2007), piperidinyl diphenylsulfonyl sulfonamide 1“WAY-316606” (Bodine et al., 2009), (hetero)arylpyrimidines (Gilbert etal., 2010), 10Z-Hymenialdisine, TCS 21311, TWS 119, GSK-3β Inhibitor II,GSK-3β Inhibitor I, GSK-3β Inhibitor XXVII, FRATtide, Cdk1/5 Inhibitorand Bikinin.

Cell culture systems comprise a cell culture solution of the inventionand an epithelial organoid, epithelial stem cell or epithelialprogenitor cell, or a population of epithelial stem cells or epithelialprogenitor cells. Epithelial organoids are known in the art (Yao et al.,2010; Lukacs et al., 2010). Epithelial stem cells include, but are notlimited to, stem cells of the intestine, stomach, lung, pancreas, andcolon. Epithelial stem cells also include LGR5 positive stem cells,derived from sources including but not limited to intestine, inner ear,brain, kidney, liver, retina, stomach, pancreas, breast, hair follicle,ovary, adrenal medulla, skin, thymus, taste buds, mammory glands,carcinomas and tumors. Epithelial stem cells also include quiescentprecursors of LGR5 positive stem cells that express LGR5 (Buczacki etal., 2013). A population of epithelial stem cells or epithelialprogenitor cells in a cell culture system can comprise, for example, atleast 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% ofthe cells in the system. Preferably, the population of epithelial stemcells or epithelial progenitor cells is maintained during repeatedpassages.

In specific embodiments, human epithelial stem cells can be cultured inthe presence of additional components including Nicotinamide or a Sirtlspecific HDAC inhibitor such as EX527.

In specific embodiments, epithelial stem cells derived from the innerear can be cultured in the presence of a Wnt agonist, a histonedeacetylase inhibitor, an Epidermal Growth Factor, a basic FibroblastGrowth Factor and optionally, a Bone Morphogenic Protein.

Cell culture systems can comprise additional components, including, butnot limited to, a submucosa base and a coating comprising collagen toform 3-dimensional tissue constructs suitable for transplantation. Thecollagen coating can be overlaid upon and/or surround the selectedepithelial tissue or cell type as well as being placed between theselected epithelial tissue or cell type and the submucosa base. Selectedepithelial tissue or cell types include, but are not limited to,epithelial stem cells, isolated tissue comprising epithelial stem cells,or epithelial organoids.

Small intestinal submucosa (SIS) is a common, biocompatible andclinically utilized scaffold (de la Fuente et al., 2003; Ueno et al.,2007; Schultz et al., 2002; Kehoe et al., 2012). Submucosal-basedscaffolds undergo rapid neovascularization, granulation, biodegradationand are generally well-conserved in terms of protein composition acrossspecies. An improved submucosal-based culture system for 3-dimensionaltissue constructs is prepared by seeding the submucosa with apreselected epithelial cell type and facilitating growth with acollagen-based overlay. Varying the composition of SIS with this overlayfacilitates cell adhesion and growth on SIS, resulting in 3-dimensionalexpansion of submucosal-adhered cells into large, epithelial organoids.Animal-derived tissue matrix scaffolds (e.g. stomach, bladder,alimentary, respiratory, genital submucosa, and liver basement membrane)from warm-blooded vertebrates are interchangeable with SIS and thuswithin the scope of this disclosure.

Tissue constructs can be cultured in the presence of cell culturesolutions known in the art or cell culture solutions of the inventiondescribed herein above. For example, tissue constructs can be culturedin the presence of a cell culture solution comprising an inhibitor of aBone Morphogenic Protein, R-spondin 1, CHIR99021 and a histonedeacetylase inhibitor. Additionally, the submucosa base can containsimilar combinations of small molecules and/or growth factors,including, but not limited to, Epidermal Growth Factor, a BoneMorphogenic Protein, R-spondin 1, CHIR99021, Y-27632 and a histonedeacetylase inhibitor.

In alternative embodiments, collagen-free epithelial cell culturesystems are provided, where the submucosa base contains combinations ofsmall molecules and/or growth factors such as Epidermal Growth Factor, aBone Morphogenic Protein, R-spondin 1, CHIR99021, Y-27632 and a histonedeacetylase inhibitor. Collagen-free tissue constructs can be culturedin the presence of cell culture solutions known in the art or asdescribed herein above.

II. Methods Employing Cell Culture Solutions and Systems

Cell culture solutions and systems of the invention can be used to formepithelial organoids from isolated epithelial stem cells with highefficiency. In a specific embodiment, incubating isolated epithelialstem cells in the presence of Noggin, R-spondin 1, CHIR99021 and ahistone deacetylase inhibitor (e.g., Valproic acid) forms epithelialcell colonies with an efficiency of at least about 25%, 35%, 40%, 50%,60%, 70%, 80%, 90% or 100%. In another specific embodiment, singleisolated epithelial stem cells incubated in the presence of Noggin,R-spondin 1, CHIR99021 and a histone deacetylase inhibitor formepithelial cell colonies with an efficiency of least about 6% to about100%.

Epithelial stem cells maintained within cell culture solutions andsystems of the invention can be subsequently directed into specificdifferentiation pathways, including those that result in the formationof paneth cells, enterocytes, goblet cells and enteroendocrine cells.

Paneth cells have been shown to be an important constituent of the Lgr5⁺stem cell niche within intestinal crypts that provide essential signalsfor stem cell maintenance (Sato et al., 2011b; Yilmaz et al., 2012).First incubating epithelial stem cells in the presence of a cell culturesolution comprising an inhibitor of a BMP, R-spondin 1, CHIR99021 and ahistone deacetylase inhibitor (e.g., valproic acid) and subsequentlyfurther incubating the epithelial stem cells in the presence of at leastone Wnt agonist and at least one inhibitor of Notch (e.g., DAPT)produces paneth cells. Likewise, subsequent further incubation of anepithelial stem cell in the presence of at least one Wnt inhibitor andat least one histone deacetylase inhibitor produces enterocytes; andsubsequent further incubation of an epithelial stem cell in the presenceof at least one Wnt inhibitor and at least one Notch inhibitor producesgoblet cells. The Wnt inhibitor can be, but is not limited to,N-(6-Methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-4-oxo-3-phenylthieno[3,2-d]pyrimidin-2-yl)thio]-acetamide(“IWP-2”) (Chen, Dodge et al. 2009). The Notch inhibitor can be, but isnot limited to N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycinet-butyl ester (“DAPT” or “LY-374973”) (Dovey. John et al. 2001),N1-[(7S)-6,7-dihydro-6-oxo-5H-dibenz[b,d]azepin-7-yl]-2,2-dimethyl-N3-(2,2,3,3,3-pentafluoropropyl)-(“RO4929097”,Propanediamide) (He, Luistro et al. 2011),(S)-2-hydroxy-3-methyl-N-((S)-1-((S)-3-methyl-2-oxo-2,3,4,5-tetrahydro-1H-benzo[d]azepin-1-ylamino)-1-oxopropan-2-yl)butanamide(“LY450139”) (Lanz, Hosley et al. 2004),N-[(1S)-2-[[(7S)-6,7-dihydro-5-methyl-6-oxo-5H-dibenz[b,d]azepin-7-yl]amino]-1-methyl-2-oxoethyl]-2-hydroxy-3-methyl-,(2S)-(“LY900009”, Butanamide) (Selleckchem: Catalog No.S7168),N-[(1S)-2-[[(7S)-6,7-dihydro-5-(2-hydroxyethyl)-6-oxo-5H-pyrido[3,2-a][3]benzazepin-7-yl]amino]-1-methyl-2-oxoethyl]-4,4,4-trifluoro-(“LY3039478”,Butanamide) Selleckchem: Catalog No.S7169,N-[(1S)-2-[[(7S)-6,7-dihydro-5-methyl-6-oxo-5H-dibenz[b,d]azepin-7-yl]amino]-1-methyl-2-oxoethyl]-3,5-difluoro-α-hydroxy-,(αS)-(“LY411575”, Benzeneacetamide) (Wehner, Cizelsky et al. 2014),7-(S)-[N′(3,5-difluorophenylacetyl)-L-alaninyl]amino-5-methyl-5,7-dihydro-6H-dibenz[b,d]azepin-6-one(“YO-01027” (DBZ)) (Milano, McKay et al. 2004),(2R)-2-(N-(2-fluoro-4-(1,2,4-oxadiazol-3-yl)benzyl)-4-chlorophenylsulfonamido)-5,5,5-trifluoropentanamide(“BMS-708163”) (Saito, Fu et al. 2014),(2R,3S)-N-[(3S)-1-Methyl-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl]-2,3-bis(3,3,3-trifluoropropyl)succinamide(“BMS-906024”) (Huang, Greer et al. 2009),(S,S)-2-[2-(3,5-Difluorophenyl)-acetylamino]-N-(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl)-propionamide(“Compound E”) (Milano, McKay et al. 2004),2-[(1R)-1-[[(4-Chlorophenyl)sulfonyl](2,5-difluorophenyl)amino]ethyl-5-fluorobenzenebutanoicacid (“BMS-299897”) (Anderson, Holtz et al. 2005), SAHM1 CalbiochemCatalogue Number: 491002, (Abeta42-Selective) Calbiochem CatalogueNumber: 565792, and N-(2-Bromophenyl)-N′-(2-hydroxy-4-nitrophenyl)urea(“SB 225002”) (Bakshi, Jin et al. 2009).

Subsequent further incubation of an epithelial stem cell in the presenceof at least of at least one inhibitor of Notch and an agent thatinhibits at least one of a Receptor Tyrosine Kinase (RTK), aMitogen-activated protein (MAP) kinase, also refered to as MAPK/ERK, oran Extracellular signal-regulated kinase (ERK), also referred to asMAPK/ERK, produces enteroendocrine cells. The MAP kinase can be, but isnot limited to, Mitogen-activated protein kinase kinase and the agentthat inhibits a MAP kinase, can be, but is not limited to,N-[(2S)-2,3-dihydroxypropyl]-3-[(2-fluoro-4-iodophenyl)amino]-4-pyridinecarboxamide(“AS-703026”) (Kim, Kong et al. 2010),N-[(2R)-2,3-Dihydroxypropoxy]-3,4-difluoro-2-[(2-fluoro-4-iodophenyl)amino]-benzamide(“PD0325901”) (Thompson and Lyons 2005),5-(2-Phenyl-pyrazolo[1,5-a]pyridin-3-yl)-1H-pyrazolo[3,4-c]pyridazin-3-ylamine(“FR 180204”) (Ohori, Kinoshita et al. 2005),2-(2-amino-3-methoxyphenyl)-4H-chromen-4-one (“PD98059”) (Alessi, Cuendaet al. 1995),6-(4-bromo-2-chlorophenylamino)-7-fluoro-N-(2-hydroxyethoxy)-3-methyl-3H-benzo[d]imidazole-5-carboxamide(“Selumetinib”) (Huynh, Soo et al. 2007),(Z)-3-amino-3-(4-aminophenylthio)-2-(2-(trifluoromethyl)phenyl)acrylonitrile(“SL-327”) (Chen, Operana et al. 2005),(2Z,3Z)-2,3-bis(amino(2-aminophenylthio)methylene)succinonitrile,ethanol(“U0126”) (Favata, Horiuchi et al. 1998),(R)-3-(2,3-dihydroxypropyl)-6-fluoro-5-(2-fluoro-4-iodophenylamino)-8-methylpyrido[2,3-d]pyrimidine-4,7(3H,8H)-dione(“TAK-733”) (Dong, Dougan et al. 2011) andN-(3-(3-cyclopropyl-5-(2-fluoro-4-iodophenylamino)-6,8-dimethyl-2,4,7-trioxo-3,4,6,7-tetrahydropyrido[4,3-d]pyrimidin-1(2H)-yl)phenyl)acetamide(“Trametinib”) (Gilmartin, Bleam et al. 2011). The agent that inhibitsan RTK can be, but is not limited to,N-(3-chloro-4-fluorophenyl)-7-methoxy-6-[3-(4-morpholinyl)propoxy]-4-quinazolinamine(“Gefitinib”) (Ciardiello 2000),(E)-2-Cyano-3-(3,4-dihydroxyphenyl)-2-propenamide (“AG 99”) (Gazit,Yaish et al.1989),4-[[(2S)-2-(3-Chlorophenyl)-2-hydroxyethyl]amino]-3-[7-methyl-5-(4-morpholinyl)-1H-benzimidazol-2-yl]-2(1H)-pyridinone(“BMS 536924”) (Huang, Greer et al. 2009),5-(2-Phenyl-pyrazolo[1,5-a]pyridin-3-yl)-1H-pyrazolo[3,4-c]pyridazin-3-ol(“FR 180209”) (Anastassiadis, Duong-Ly et al. 2013),N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazolin-4-aminehydrochloride (“Erlotinib”) (Kuiper, Heideman et al. 2014),(S,E)-N-(4-(3-chloro-4-fluorophenylamino)-7-(tetrahydrofuran-3-yloxy)quinazolin-6-yl)-4-(dimethylamino)but-2-enamide(“Afatinib”) (Minkovsky and Berezov 2008),N-(4-(3-fluorobenzyloxy)-3-chlorophenyl)-6-(5-((2-(methylsulfonyl)ethylamino)methyl)furan-2-yl)quinazolin-4-amine,di4-methylbenzenesulfonate(“Lapatinib”) (Xia, Mullin et al. 2002),N-(3-(5-chloro-2-(2-methoxy-4-(4-methylpiperazin-1-yl)phenylamino)pyrimidin-4-yloxy)phenyl)acrylamide(“WZ4002”) (Sakuma, Yamazaki et al. 2012) and2-[(3,4-dihydroxyphenyl)methylene]-(“AG-18”, Propanedinitrile) (Gazit,Yaish et al. 1989).

Cell culture solutions and systems of the invention can additionally beused to form 3-dimensional tissue constructs comprising transplantableepithelium for regenerative purposes. Such tissue constructs can betransplanted into hosts according to methods known in the art (Lloyd etal. 2006; Gupta et al. 2006; Yui et al. 2012). Tissues that aresusceptible to treatment include all damaged tissues, including thosewhich may have been damaged by disease, injury, trauma, an autoimmunereaction, or by a viral or bacterial infection. Minimally-invasivetransplant techniques can be employed, including image-guidedtechnology. Tissue constructs can be injected or implanted directly intothe damaged tissue, where they may multiply and eventually differentiateinto the required cell type, in accordance with their location in thebody. Tissue constructs may be directly implanted or injected viacolonic enema. Micronization can be employed prior to oral delivery forupper intestinal applications. Accordingly, damaged tissues particularlywell suited for repair include tissues of the colon, small intestine,pancreas, esophagus and gastric system. The skilled person will be awarewhat the appropriate dosage of tissue constructs will be for aparticular condition to be treated.

Cell culture solutions and systems of the invention can additionally beused to predict the efficacy of a chemotherapeutic agent, orcombinations of chemotherapeutic agents, in vivo. Such methods areparticularly relevant to use in clinical settings since many patientsare treated with multiple drugs.

Tumor organoids can be formed according to methods known in the art byculturing isolated tumor cell aggregates or single cells in culturesolutions of the invention (Sato et al., 2011a). Such cultures can beused as clinical models for various cancers, including but not limitedto, prostate cancer, breast cancer, gastric cancer, pancreatic cancer,lung cancer, brain cancer, colon cancer, intestinal cancer and bladdercancer.

Tumor organoids can be incubated in the presence of a cell culturesolution of the invention (e.g., comprising an inhibitor of a BMP,R-spondin 1, a Wnt agonist, a histone deacetylase inhibitor) and achemotherapeutic agent(s). Next, a relevant parameter is measured andevaluated. Relevant parameters include inhibition of cell viability,inhibition cell proliferation, inhibition of tumor associated geneexpression, activation of apoptosis and inhibition of cell survival.Detecting an increase in the parameter compared to a reference (e.g.,control) indicates efficacy of the chemotherapeutic agent relative tothe tumor organoid, which is predictive of efficacy of thechemotherapeutic agent(s) in vivo.

In general, chemotherapeutic agents are incubated with the cell culturesystem comprising tumor organoids in a dosage range estimated to betherapeutic and for a duration sufficient to produce a physiologicaleffect. The incubation time can range between about 1 hour to 24 hours,or can be extended as necessary for several days or even weeks. Theincubation conditions typically involve using culture solutions of theinvention and maintaining temperatures of about 37° C.

A chemotherapeutic agent is any substance that is evaluated for itsability to cure, mitigate, treat, or prevent cancer in a subject andincludes, but is not limited to, a chemical compound, biologic agent,protein, peptide, nucleic acid, lipid, polysaccharide, supplement, andan antibody.

Inhibition of tumor associated gene expression can be determinedaccording to methods known in the art. For example, inhibition of tumorassociated gene expression relative to a control can be detected bymicrochip analysis, RT-PCR, in situ hybridization, fluorescence in situhybridization or Northern analysis. Inhibition of tumor associatedprotein expression relative to a control can be detected by quantitativeWestern blot, immunohistochemistry, immunofluorescence, enzyme-linkedimmunosorbent assay, amino acid sequence analysis, fluorescenceactivated cell sorting or protein concentration assays. For example, agastric cancer gene screening assay can be utilized to identify changesin gene expression for angiotensin, apolipoprotein E, apolipoproteinA-I, ceruloplasmin, prothrombin, fibronectin, vitamin D-binding protein,gelsolin, inter-alpha-trypsin inhibitor heavy chain H3, kininogen-1,serum paraoxonase/arylesterase 1, alpha-1-antichymotrypsin andtransthyretin.

Activation of apoptosis can be determined according to methods known inthe art. For example, increases in cell death relative to a control canbe detected by lactate dehydrogenase release, caspase activity, annexinV staining, phosphatidylserine staining or TUNEL assay. Certain assaysdetect comparatively late events in the process of cell death, such aslactate dehydrogenase release. Caspase activation is a common feature ofchronic toxicity and cell death. Caspase activity can be measuredrelatively quickly after a toxic insult (30 minutes to 4 hours) byfluorescence spectroscopy, thus lending itself to high-throughputscreening techniques. Other markers and assays commonly used to monitorapoptosis or necrosis of cells can include, but are not limited to, thepresence of phosphatidylserine on the outer leaflet of the plasmamembrane of affected cells, annexin V staining, and terminaldeoxynucleotidyltransferase nick-end labeling assay (TUNEL).

Inhibition of cell viability can be determined according to methodsknown in the art, including, but not limited to, differential countingof viable and dead cells using vital dyes, such as trypan blue,4,6-diaminophenylindole (DAPI), and propidium iodide.

Inhibition of cell proliferation can be determined according to methodsknown in the art, including, but not limited to quantification of DNAvia bromodeoxyuridine incorporation, measuring tritiated thymidine(3H-thymidine), propidium iodide staining, intracellular metabolicanalysis via tetrazolium salt or AlamarBlue reduction and quantitationof intracellular ATP concentration. Further methods include directmeasuring total nuclei acid content of lysed cells viaspectrophotometric analysis; fluorescent tagging with anti-cdc6-PeptideAntibody, anti-Human mRNA-Binding Protein HuR Antibody (Anti-HuRAntibody), antibodies against D Cyclins and Cyclin-Dependent KinaseInhibitors; Ki-67 antigen detection; measuring protein content viaquantitative Western blot, immunohistochemistry, immunofluorescence,enzyme-linked immunosorbent assay, amino acid sequence analysis,fluorescence activated cell sorting or protein concentration assays.Commercially available kits employing the above methods includeChromaTide™ nucleotide labelling, Succinimidyl ester ofcarboxyfluorescein diacetate, ABSOLUTE-S™ SBIP Cell Proliferation AssayKit, Vybrant DiI Cell-Labeling Solution, CyQUANT Cell ProliferationAssay Kit, Vybrant™ MTT Cell Proliferation Assay Kit, and FluoReporter™Blue Fluorometric nucleic acid assay kit.

Suppression of cell survival can be determined according to methodsknown in the art, including clonogenic assays.

III. Methods of Promoting Expansion of Epithelial Cells or Growth ofEpithelial Tissues In Vivo

Epithelial stem cells, including stem cells of the intestine, stomach,lung, pancreas and colon and in particular, LGR5 positive stem cellspresent within intestine, inner ear, brain, kidney, liver, retina,stomach, pancreas, breast, hair follicle, ovary, adrenal medulla, skin,thymus, taste buds, and mammory glands, can be expanded in vivo throughadministration of a Wnt agonist and a histone deacetylase inhibitor or aWnt agonist and a Notch agonist to a subject. These combinations promoteexpansion of epithelial cells resulting in growth of epithelial tissuesin vivo.

In specific embodiments, intestinal epithelial cells can be formed invivo following administration of a Wnt agonist, e.g., CHIR99021 and ahistone deacetylase inhibitor, e.g., Valproic acid, or a Wnt agonist,e.g., CHIR99021 and a Notch agonist to a subject.

In some embodiments, these combinations, e.g., CHIR99021 and Valproicacid, can treat intestinal disorders in a subject, including, but notlimited, to enterocolitis; viral infections, such as non-specificenteritis or specific viral enteritis; diverticulitis; bacterialenterocolitis, such as salmonellosis, shigellosis, campylobacterenterocolitis, or yersinia enterocolitis; protozoan infections such asamebiasis; helminthic infection; and pseudomembraneous colitis andpulmonary complications in cystic fibrosis and chronic obstructivepulmonary disease; appendicitis; atrophic gastritis; Barrett'sesophagus; pneumonitis; cervicitis; chronic interstitial nephritis;colitis; colonic diverticulitis; conjunctivitis; contact dermatitis;Curling's ulcers; Cushing's ulcers; cystitis; gangrene; gingivitis;mastitis; esophagitis; pancreatitis; panniculitis; phlegmonousgastritis; glomerulonephritis; and autoimmune diseases including, butnot limited to, inflammatory bowel disease, ulcerative colitis, Crohn'sdisease, Addison's disease and glomerulonephritis (e.g., crescenticglomerulonephritis, proliferative glomerulonephritis).

The dosage administered will be dependent upon the age, sex, health, andweight of the recipient, kind of concurrent treatment, if any, frequencyof treatment and the nature of the effect desired. The dose ranges forthe administration of the compositions of the present invention arethose large enough to produce the desired effect. The doses should notbe so large as to cause adverse side effects, such as unwantedcross-reactions, anaphylactic reactions, and the like. Generally, thedosage will vary with the condition and extent of the disease in thepatient. Counter indications, if any, immune tolerance, and othervariables will also affect the proper dosage. For instance, taking intoaccount such factors as the age, weight, sex, species, generalhealth/condition of the patient, the condition to be treated, timing oftreatments, the LD50 of the active ingredient involved in a suitableanimal model (e.g., rodent, mice), and other known factors; and suchdosages can be on the order of micrograms to milligrams such as on theorder of 0.5 to 500 mg/kg, or another suitable amount, or can becomputed from Examples herein, e.g., considering the average weight of atypical test animal (such as mice) and the dosages administered thereto(e.g., 100 micrograms), and thus the skilled artisan can determinedosages without undue experimentation. In particular, in human subjects,CHIR99021 is administered in an amount of about 0.1 mg/kg/day to about100 mg/kg/day and the amount of Valproic acid is administered in anamount of about lmg/kg/day to about 1000 mg/kg/day. In specificembodiments, the amount of Valproic acid is 15-40 mg/kg/day.

The pharmaceutical compositions of CHIR99021 and Valproic acid can beconcomitantly or sequentially administered by any means that achievetheir intended purpose. For example, administration can be by topical,parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal,transdermal, rectal or buccal routes. Alternatively, or concurrently,administration can be by the oral route. From the foregoing description,it will be apparent that variations and modifications may be made to theinvention described herein to adopt it to various usages and conditions.Methods and materials are described herein for use in the presentinvention; other, suitable methods and materials known in the art canalso be used. The materials, methods, and examples are illustrative onlyand not intended to be limiting. Such embodiments are also within thescope of the following claims. The recitation of a listing of elementsin any definition of a variable herein includes definitions of thatvariable as any single element or combination (or subcombination) oflisted elements. The recitation of an embodiment herein includes thatembodiment as any single embodiment or in combination with any otherembodiments or portions thereof. All publications, patent applications,patents, sequences, database entries, and other references mentionedherein are incorporated by reference in their entirety.

Exemplary embodiments of the invention can also be described by any oneof the following numbered paragraphs:

1. A method of forming an enterocyte in a cell culture system comprisingincubating an epithelial stem cell in the presence of at least one Wntinhibitor and at least one histone deacetylase inhibitor, each in anamount sufficient to produce an enterocyte in a cell culture system.

2. The method of paragraph 1, wherein the histone deacetylase inhibitoris a Pan-HDAC inhibitor.

3. The method of paragraph 2, wherein the Pan-HDAC inhibitor is selectedfrom the group consisting of Valproic acid, Trichostatin A,suberoylanilide hydroxamic acid and SBHA.

4. The method of paragraph 1, wherein the histone deacetylase inhibitoris an HDAC6 inhibitor.

5. The method of paragraph 4, wherein the HDAC6 inhibitor is selectedfrom the group consisting of Tubacin, Tubastatin A and Compound 7.

6. The method of paragraph 1, wherein the Wnt inhibitor is selected fromthe group consisting of IWP-2, XAV-939, ICG-001, LGK-974, IWR-1-endo,KY02111, Wnt-059, DKK-1, FH-535, Box5, Peptide Pen-N3, Anti-SFRPantibody, and Anti-LRP6 antibody.

7. The method of paragraph 1, further comprising incubating theepithelial stem cell in the presence of an inhibitor of a BoneMorphogenic Protein.

8. The method of paragraph 7, wherein the Bone Morphogenic Protein isselected from the group consisting of Noggin, Chordin, Follistatin, DAN,proteins comprising a DAN cysteine-knot domain, Sclerostin, TwistedGastrulation, Uterine Sensitivity-Associated Gene-1, Connective-TissueGrowth Factor, Inhibin, BMP-3, and Dorsomorphin.

9. The method of paragraph 1, further comprising incubating theepithelial stem cell in the presence of an Epidermal Growth Factor.

10. A method of forming a goblet cell in a cell culture systemcomprising incubating an epithelial stem cell in the presence of atleast one Wnt inhibitor and at least one Notch inhibitor, each in anamount sufficient to produce a goblet cell in a cell culture system.

11. The method of paragraph 10, wherein the Notch inhibitor is selectedfrom the group consisting of DAPT, RO4929097, LY450139, LY900009,LY3039478, LY411575, YO-01027, BMS-708163, BMS-906024, Compound E,BMS-299897, SAHM1, Abeta42-Selective and SB 225002.

12. The method of paragraph 10, wherein the Wnt inhibitor is selectedfrom the group consisting of IWP-2, XAV-939, ICG-001, LGK-974,IWR-1-endo, KY02111, Wnt-059, DKK-1, FH-535, Box5, Peptide Pen-N3,Anti-SFRP antibody, Anti-LRP6 andtibody, and Anti-APC antibody.

13. The method of paragraph 10, further comprising incubating theepithelial stem cell in the presence of an Epidermal Growth Factor.

14. A method of forming an enteroendocrine cell in a culture systemcomprising incubating an epithelial stem cell in the presence of atleast one inhibitor of Notch and an agent that inhibits at least one ofa Receptor Tyrosine Kinase, a Mitogen-activated protein (MAP) kinase oran Extracellular signal-regulated kinase (ERK), each in an amountsufficient to produce an enteroendocrine cell in a cell culture system.

15. The method of paragraph 14, wherein the Notch inhibitor is selectedfrom the group consisting of DAPT, RO4929097, LY450139, LY900009,LY3039478, LY411575, YO-01027, BMS-708163, BMS-906024, Compound E,BMS-299897, SAHM1, Abeta42-Selective and SB 225002.

16. The method of paragraph 14, wherein the MAP kinase isMitogen-activated protein kinase kinase (MEK).

17. The method of paragraph 14, wherein the agent that inhibits a MAPkinase is selected from the group consisting of AS-703026, PD0325901,PD98059, Selumetinib, SL-327, U0126, TAK-733 and Trametinib.

18. The method of paragraph 14, wherein the agent that inhibits an RTKis selected from the group consisting of Gefitinib, AG 99, Erlotinib,Afatinib, Lapatinib, WZ4002 and AG-18.

19. The method of paragraph 14, wherein the agent that inhibits an ERKis AS-703026 or PD0325901.

20. The method of paragraph 14, further comprising incubating theepithelial stem cell in the presence of an inhibitor of a BoneMorphogenic Protein.

21. The method of paragraph 20, wherein the Bone Morphogenic Protein isselected from the group consisting of Noggin, Chordin, Follistatin, DAN,proteins comprising a DAN cysteine-knot domain, Sclerostin, TwistedGastrulation, Uterine Sensitivity-Associated Gene-1, Connective-TissueGrowth Factor, Inhibin, BMP-3, and Dorsomorphin.

22. The method of paragraph 14, further comprising incubating theepithelial stem cell in the presence of an agent that binds to theLeucine-rich repeat-containing G-protein coupled receptor 5.

23. The method of paragraph 22, wherein the agent that binds to theLeucine-rich repeat-containing G-protein coupled receptor 5 is selectedfrom the group consisting of R-spondin 1, R-spondin 2, R-spondin 3 andR-spondin 4

24. The method of paragraph 14, further comprising incubating theepithelial stem cell in the presence of an Epidermal Growth Factor.

25. A method of forming intestinal epithelial cells in a subject in needthereof, comprising administering to the subject a Wnt agonist and ahistone deacetylase inhibitor in an amount sufficient to form intestinalepithelial cells in the subject.

26. The method of paragraph 25, wherein the subject is a human.

27. The method of paragraph 25, wherein the Wnt agonist is selected fromthe group consisting of: Wnt-1/Int-1, Wnt-2/Irp (Int-I-related Protein),Wnt-2b/13, Wnt-3/Int-4, Wnt-3a, Wnt-4, Wnt-5a, Wnt-5b, Wnt-6, Wnt-7a,Wnt-7b, Wnt-8a/8d, Wnt-8b, Wnt-9a/14, Wnt-9b/14b/15, Wnt-10a,Wnt-10b/12, Wnt-11, Wnt-16, R-spondin 1, R-spondin 2, R-spondin 3,R-spondin 4, Norrin, CHIR99021, LiCl, BIO((2′Z,3′E)-6-Bromoindirubin-3′-oxime), CHIR98014, SB 216763, SB 415286,3F8, Kenpaullone, 1-Azakenpaullone, TC-G 24, TCS 2002, AR-A 014418,2-amino-4-[3,4-(methylenedioxy)benzyl-amino]-6-(3-methoxyphenyl)pyrimidine,IQ 1, DCA, QS 11, WAY-316606, (hetero)arylpyrimidines,10Z-Hymenialdisine, TCS 21311, TWS 119, GSK-3 Inhibitor IX, GSK-3Inhibitor IV, GSK-3β Inhibitor II, GSK-3β Inhibitor I, GSK-3β InhibitorXXVII, GSK-3beta Inhibitor XXVI, FRATtide, Cdk1/5 Inhibitor, Bikinin,and 1-Azakenpaullone.

28. The method of paragraph 25, wherein the histone deacetylaseinhibitor is a Pan-HDAC inhibitor.

29. The method of paragraph 28, wherein the Pan-HDAC inhibitor isselected from the group consisting of Valproic acid, Trichostatin A,suberoylanilide hydroxamic acid and SBHA.

30. The method of paragraph 25, wherein the histone deacetylaseinhibitor is an HDAC6 inhibitor.

31. The method of paragraph 30, wherein the HDAC6 inhibitor is selectedfrom the group consisting of Tubacin, Tubastatin A and Compound 7.

32. The method of paragraph 25, wherein the Wnt agonist is CHIR99021 andthe histone deacetylase inhibitor is Valproic acid.

33. The method of paragraph 32, wherein the CHIR99021 is administered inan amount of about 0.1 mg/kg/day to about 100 mg/kg/day and the Valproicacid is administered in an amount of about 1 mg/kg/day to about 1000mg/kg/day.

34. A method of generating epithelial tissue in a subject in needthereof, comprising administering a Wnt agonist and a histonedeacetylase inhibitor or a Wnt agonist and a Notch agonist to thesubject in an amount sufficient to increase epithelial stem cells withinthe epithelial tissue, thereby generating epithelial tissue in thesubject.

35. The method of paragraph 34, wherein the epithelial stem cell is aLGR5 positive stem cell present within intestine, inner ear, brain,kidney, liver, retina, stomach, pancreas, breast, hair follicle, ovary,adrenal medulla, skin, thymus, taste buds or mammary glands.

36. A method of forming intestinal epithelial cells in a subject in needthereof, comprising administering to the subject a Wnt agonist and aNotch agonist in an amount sufficient to form intestinal epithelialcells in the subject.

35. The method of paragraph 34 or 36, wherein the subject is a human.36. The method of paragraph 34 or 36, wherein the Wnt agonist isselected from the group consisting of: Wnt-1/Int-1, Wnt-2/Irp(Int-I-related Protein), Wnt-2b/13, Wnt-3/Int-4, Wnt-3a, Wnt-4, Wnt-5a,Wnt-5b, Wnt-6, Wnt-7a, Wnt-7b, Wnt-8a/8d, Wnt-8b, Wnt-9a/14,Wnt-9b/14b/15, Wnt-10a, Wnt-10b/12, Wnt-11, Wnt-16, R-spondin 1,R-spondin 2, R-spondin 3, R-spondin 4, Norrin, CHIR99021, LiCl, BIO((2′Z,3′E)-6-Bromoindirubin-3′-oxime), CHIR98014, SB 216763, SB 415286,3F8, Kenpaullone, 1-Azakenpaullone, TC-G 24, TCS 2002, AR-A 014418,2-amino-4-[3,4-(methylenedioxy)benzyl-amino]-6-(3-methoxyphenyl)pyrimidine,IQ 1, DCA, QS 11, WAY-316606, (hetero)arylpyrimidines,10Z-Hymenialdisine, TCS 21311, TWS 119, GSK-3 Inhibitor IX, GSK-3Inhibitor IV, GSK-3β Inhibitor II, GSK-3β Inhibitor I, GSK-3β InhibitorXXVII, GSK-3beta Inhibitor XXVI, FRATtide, Cdkl/5 Inhibitor, Bikinin,and 1-Azakenpaullone.

37. The method of paragraph 34 or 36, wherein the Notch agonist is aNotch1 antibody (N1 Ab), Delta 1, Delta-like 3, Delta-like 4, Jagged 1,Jagged 2, DSL peptide and Delta D.

38. A method of treating an intestinal disorder, the method comprisingadministering to the subject a Wnt agonist and a histone deacetylaseinhibitor or a Wnt agonist and a Notch.

39. The method of paragraph 38, wherein the subject is a human.

40. The method of paragraph 38, wherein the Wnt agonist is selected fromthe group consisting of: Wnt-1/Int-1, Wnt-2/Irp (Int-I-related Protein),Wnt-2b/13, Wnt-3/Int-4, Wnt-3a, Wnt-4, Wnt-5a, Wnt-5b, Wnt-6, Wnt-7a,Wnt-7b, Wnt-8a/8d, Wnt-8b, Wnt-9a/14, Wnt-9b/14b/15, Wnt-10a,Wnt-10b/12, Wnt-11, Wnt-16, R-spondin 1, R-spondin 2, R-spondin 3,R-spondin 4, Norrin, CHIR99021, LiCl, BIO((2′Z,3′E)-6-Bromoindirubin-3′-oxime), CHIR98014, SB 216763, SB 415286,3F8, Kenpaullone, 1-Azakenpaullone, TC-G 24, TCS 2002, AR-A 014418,2-amino-4-[3,4-(methylenedioxy)benzyl-amino]-6-(3-methoxyphenyl)pyrimidine,IQ 1, DCA, QS 11, WAY-316606, (hetero)arylpyrimidines,10Z-Hymenialdisine, TCS 21311, TWS 119, GSK-3 Inhibitor IX, GSK-3Inhibitor IV, GSK-3β Inhibitor II, GSK-3β Inhibitor I, GSK-3β InhibitorXXVII, GSK-3beta Inhibitor XXVI, FRATtide, Cdk1/5 Inhibitor, Bikinin,and 1-Azakenpaullone.

41. The method of paragraph 38, wherein the histone deacetylaseinhibitor is a Pan-HDAC inhibitor.

42. The method of paragraph 41, wherein the Pan-HDAC inhibitor isselected from the group consisting of Valproic acid, Trichostatin A,suberoylanilide hydroxamic acid and SBHA.

43. The method of paragraph 38, wherein the histone deacetylaseinhibitor is an HDAC6 inhibitor.

44. The method of paragraph 43, wherein the HDAC6 inhibitor is selectedfrom the group consisting of Tubacin, Tubastatin A and Compound 7.

45. The method of paragraph 38, wherein the Wnt agonist is CHIR99021 andthe histone deacetylase inhibitor is Valproic acid.

46. The method of paragraph 45, wherein the CHIR99021 is administered inan amount of about 0.1 mg/kg/day to about 100 mg/kg/day and the Valproicacid is administered in an amount of about lmg/kg/day to about 1000mg/kg/day.

47. The method of paragraph 38, wherein the Notch agonist is a Notch1antibody (N1 Ab), Delta 1, Delta-like 3, Delta-like 4, Jagged 1, Jagged2, DSL peptide and Delta D.

48. The method of any of paragraphs 38-47, wherein the intestinaldisorder is selected from the group consisting of:

-   -   enterocolitis; viral infections, such as non-specific enteritis        or specific viral enteritis;    -   diverticulitis; bacterial enterocolitis, such as salmonellosis,        shigellosis, campylobacter enterocolitis, or yersinia        enterocolitis; protozoan infections such as amebiasis;        helminthic infection; and pseudomembraneous colitis and        pulmonary complications in cystic fibrosis and chronic        obstructive pulmonary disease; appendicitis; atrophic gastritis;        Barrett's esophagus; pneumonitis; cervicitis; chronic        interstitial nephritis; colitis; colonic diverticulitis;        conjunctivitis; contact dermatitis; Curling's ulcers; Cushing's        ulcers;    -   cystitis; gangrene; gingivitis; mastitis; esophagitis;        pancreatitis; panniculitis;    -   phlegmonous gastritis; glomerulonephritis; and autoimmune        diseases including, but not limited to, inflammatory bowel        disease, ulcerative colitis, Crohn's disease, Addison's disease        and glomerulonephritis (e.g., crescentic glomerulonephritis,        proliferative glomerulonephritis).

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

EXAMPLES Example 1 Self-Renewal of Lgr5⁺ Intestinal Stem Cells isMaintained Using a Combination of Small Molecules

The self-renewal and differentiation of ISC is controlled by thecoordinated regulation of several signaling pathways (Crosnier,Stamataki, & Lewis, 2006; Scoville, Sato, He, & Li, 2008; van der Flier& Clevers, 2009). In this study small molecules were identified thattarget relevant signaling pathways to maintain the self-renewal statusof Lgr5⁺ stem cells and to control their differentiation independentlyof cues provided by other cell types.

Crypts and single Lgr5-GFP cells were isolated as previously described(Sato et al., 2009). Briefly, the proximal half of the small intestinewas harvested, opened longitudinally, and washed with cold PBS to removeluminal content. The tissue was then cut into 2-4 mm pieces withscissors and further washed 5-10 times with cold PBS by pipetting up anddown using a 10-ml pipette. Tissue fragments were incubated with 2 mMEDTA in PBS for 30 min on ice. After removal of EDTA, tissue fragmentswere washed with PBS to release crypts. Supernatant fractions enrichedin crypts were collected, passed through a 70 μm cell strainer andcentrifuged at 300 g for 5 minutes. The cell pellet was re-suspendedwith cell culture media without growth factors and centrifuged at 150 gto remove single cells. Crypts were then cultured or used for singlecell isolation. To obtain single cells, crypts were incubated in culturemedium for 45 minutes at 37° C. and triturated with a glass pipette.Dissociated cells were passed through 20 μm cell strainer, negativestained with propidium iodide and single viable GFP-high cells weresorted by flow cytometry (FACS Aria, BD) as previously described (Satoet al., 2009). Small intestinal crypts isolated fromLgr5-EGFP-ires-CreERT2 mice were embedded in Matrigel and cultured underconventional culture conditions in the presence of EGF, Noggin andR-spondin 1 (collectively referred to as ENR) leading to organoids withcrypts and villus-like domains and GFP⁺ cells at the crypt tips,consistent with previous reports (Sato et al., 2009). Isolated crypts orsingle cells were cultured as previously described (Sato et al., 2009)with minimal modification. Briefly, crypts or single cells wereentrapped in Matrigel and plated at the center of wells in a 24-wellplate. After polymerization of Matrigel (growth factor reduced; BDBioscience), 500 μl of culture medium (Advanced DMEM/F12 (LifeTechnologies)) was added containing growth factors including EGF (50ng/ml, Life Technologies), Noggin (100 ng/ml, Peprotech) and R-spondin 1(500 ng/ml, R&D) and small molecules including CHIR99021 (3 μM,Stemgent) and Valproic Acid (1 mM, Sigma-Aldrich). For comparison ofdifferent culture conditions, small molecules or growth factors wereadded to freshly isolated crypts immediately after plating in Matrigelto test the ability to minimize potential differentiation of the ISCwithin the crypts and thus sustain crypt cultures. Cell culture mediawas changed every other day. For single cell culture, cells wereembedded in Matrigel containing Jagged-1 peptide (1 μM; AnaSpec) andY-27632 (10 μM; Tocris) was added for the first 2 days. Cells werepassaged either as cell colonies as previously described (Sato et al.,2009) or as single cells. For single cell passage, cell culture mediumwas removed and Accutase (Life Technologies) was added. After incubationat 37° C. for 10-20 minutes, cell colonies were dissociated into singlecells by pipetting. Cells were then washed, embedded in fresh Matrigeland plated into 24-well plates. Cells cultured in the CV condition werepassaged every 6 days at a 1:20 split ratio. Approximately half of thecultured crypts contained GFP+ cells, which is consistent with the invivo GFP expression of Lgr5-GFP mice (FIG. 1).

The growth factors used in the ENR condition provide essential, but notadequate cues to sustain the self-renewal of Lgr5⁺ stem cells. Toidentify factors essential to maintaining the self-renewal status ofintestinal stem cells, selected small molecules that modulate signalingpathways of ISCs, such as Wnt, Notch, and BMP, were tested under the ENRcondition, using the Lgr5-GFP reporter. CHIR99021 (referred to herein asCHIR or C), a GSK3β inhibitor which activates the Wnt signaling pathway,promoted the proliferation of crypt cells, as indicated by quantifyingthe average size of organoids and cell numbers in the culture (FIGS. 2A, 2B and 3A, 3B). CHIR increased the percentage and relative GFPintensity of GFP⁺ cells in the culture, indicating increasedself-renewal of stem cells (FIGS. 2A and 2B). Notably, a large number ofGFP negative cells still existed in the organoids (FIG. 2A), which waslikely the result of insufficient maintenance of stem cell self-renewalor the result of promoting the proliferation of more mature GFP negativecells in the crypts. Valproic Acid (VPA or V), a histone deacetylaseinhibitor, also significantly increased the GFP expression of GFP⁺organoids, with minimal presence of GFP negative cells (FIG. 2A).Interestingly, when CHIR and VPA were combined (CV), cell proliferationas well as the percentage and relative GFP intensity of GFP expressingcells in the culture significantly increased (FIGS. 2A and 2B), withnearly pure GFP+ cells in GFP⁺ organoids (FIG. 2A), indicating minimaldifferentiation or proliferation of differentiated cells and increasedself-renewal of stem cells in this culture condition.

The GFP⁺ cells in the CV condition showed a single GFP-high populationthat corresponded to that of freshly isolated single cells (FIG. 3C),representing the Lgr5+ stem cell population as previously reported (Satoet al., 2009). Notably, in the CV condition, R-spondin 1 and Noggin werestill required to maintain the self-renewal of Lgr5+ stem cells, whileEGF promoted the proliferation of crypts, it could be removed from theculture without impacting maintenance of the Lgr5+ cells (FIG. 3D).Increasing the concentration of CHIR further eliminated the need ofR-Spondin1 to promote GFP expression (FIG. 3E), consistent with the roleof R-spondin 1 to increase Wnt/β-catenin signaling. Furthermore, VPA orCHIR+VPA also promoted GFP expression of Lgr5+ stem cells from the colon(FIG. 3F). In addition, R-Spondin 2 showed better efficacy at lowerconcentrations in promoting organoid formation in the ENR conditioncompared to R-spondin 1 (FIG. 3G). We also tested culture conditionsthat were previously shown to maintain human EPHB2⁺ colonic stem cellsor colonic crypts in a largely undifferentiated state (Jung et al.,2011; Sato et al., 2011a) but failed to achieve similar effects on smallintestinal Lgr5-GFP stem cells (FIGS. 4A and 4B), suggesting that thesefactors may act through different mechanisms on EPHB2⁺ colonic stemcells versus Lgr5+ stem cells.

To further confirm the proliferation and Lgr5⁺ self-renewal effects ofCHIR and VPA in the absence of mature cell types and GFP negative stemcells (given that crypts show a mosaic GFP expression pattern), singleGFP-high cells were isolated by FACS sorting (FIG. 3C) and cultured inMatrigel in the presence of ENR and CHIR or VPA, or in the presence ofboth compounds (CV condition). The Rho kinase inhibitor Y-27632, whichinhibits anoikis of single stem cells (Watanabe et al., 2007), was addedfor the first two days as previously described (Sato et al., 2009).Following a 7 day culture, colonies containing GFP⁺ stem cellsspontaneously formed. Similar to crypt cultures, CHIR significantlyincreased cell proliferation while it only moderately increased GFPexpression, while VPA promoted GFP expression with a minimalpro-proliferative effect. For the CV condition, cell proliferationsignificantly increased and greater than 97% of the cells in the culturewere GFP⁺ cells (FIGS. 2C-2E and 5A). It is noteworthy that compared tothe crypt cultures, when pure single Lgr5+ stem cells were cultured inCHIR, the organoids that formed contained large numbers of GFP negativecells, indicating that stem cells differentiated in this condition andthus other factors were required to maintain the self-renewal status ofLgr5⁺ stem cells.

When isolated single Lgr5-GFP⁺ cells were cultured in the standard ENRcondition, few cells grew into organoids, which is consistent withprevious reports (Sato et al., 2009) and likely due to sub-optimalculture conditions. When CHIR was added to the culture (ENR-C), thecolony-forming efficiency was significantly increased by 20-50 fold(FIGS. 2F, 2G and 5B, 5C), providing a similar response to the additionof Wnt3A when added at 100 ng/ml (FIG. 2F and Sato et al., 2011b). Insharp contrast to this, VPA only weakly increased colony-formingefficiency in the absence of CHIR (ENR-V, FIGS. 2F, 2G and 5B, 5C).Surprisingly, when isolated single Lgr5-GFP⁺ stem cells were cultured inthe presence of both CHIR and VPA, there was a synergistic effect and˜25%-40% of the total cell population grew into colonies (FIG. 2F). Thisis believed to represent the most efficient colony formation that hasbeen reported for Lgr5⁺ stem cells.

TABLE 1 Colony Numbers for Colony Formation in FIG. 2G ENR ENR--C ENR-VENR-CV ENR-W ENR-WV Average 7.333333 158.6667 32.33333 956 135.3333475.3333

TABLE 2 Colony Numbers for Colony Formation Efficiency in FIG. 5C ENRENR-C ENR-W ENR-V ENR-CV Average 3 164.75 56.25 24.5 495.25

Given that a portion of the cells that were sorted via FACS were under apro-apoptotic status and typically died within 12 hours (Sato et al.,2011b), live cells were manually counted 12 hours after seeding. Greaterthan 90% of the live cells grew into organoids when both CHIR and VPAwere present in the culture media (FIG. 5D).

TABLE 3 Colony Forming Efficiency in FIG. 5D ENR ENR-V ENR-C ENR-CVENR-W ENR-WV Average 0 0 0.142291 0.921154 0.132576 0.190111 Note: <100cells were plated so the calculated efficiency for R or RV is 0

Furthermore, cells cultured in the CV condition could be passaged assingle cells for more than 10 passages with similar colony-formingefficiency with that of freshly isolated Lgr5-GFP⁺ cells, and withoutloss of proliferative ability, and they showed normal karyotype (2n=40)(FIG. 2H). These results suggest that CHIR and VPA provide signals thatare not present in the standard ENR condition to maintain theself-renewal of Lgr5⁺ stem cells.

As previously reported, cells in the ENR condition grew into organoidswith crypt-villus structure containing all intestinal epithelial celltypes, confirmed by staining of alkaline phosphatase (Alp) positiveenterocytes, Mucin 2 (Muc2) positive goblet cells, Chromogranin A (ChgA)positive enteroendocrine cells, Lysozyme (Lyz) positive Paneth cells andLgr5-GFP⁺ stem cells. Lgr5⁺ stem cells only reside at the tips of crypt(FIGS. 6A and 7A). Ki67 and EdU staining revealed that proliferatingcells were only existent within the crypt domains (FIGS. 6B and 6C). Inthe CV condition, however, GFP⁺ stem cells were present throughout theentire colony with minimal presence of Paneth cells (FIG. 6A) andwithout the presence of other cells types. Compared to ENR culture, Ki67or EdU positive proliferating cells in the CV condition existedthroughout the cell colony (FIGS. 6B and 6C). This was confirmed withquantitative Real-time PCR whereby cells in the CV condition expressedminimal levels of Alpi (enterocytes), Muc2 (goblet cells), ChgA(enteroendocrine cells), moderate levels of Lysozyme (Paneth cells) andhigh levels of Lgr5 (ISC) compared to cells in the ENR condition (FIG.6D). This expression pattern was maintained over multiple passages, andthe Lgr5 expression level was maintained (FIG. 6D).

CHIR alone reduces enterocyte differentiation, but simultaneouslyincreased Paneth cell differentiation (FIG. 6D), which is consistentwith previous report (Farin et al., 2012). While VPA alone decreasedsecretory differentiation (FIG. 6D) and helped to maintain a higherfraction of GFP+ stem cells, it is not sufficient to suppress thedifferentiation of stem cells. Indeed, when isolated single stem cellswere cultured in the presence of VPA but without CHIR or other agentsthat promote Wnt signaling, their survival was much lower than when Wntwas present. When the Wnt pathway is blocked by IWP-2, VPA alone cannotmaintain the self-renewal of stem cells (the IV condition in FIGS. 7B,7C). The combination of CHIR and VPA suppressed both enterocyte andsecretory differentiation and maintained the self-renewal program ofLgr5⁺ stem cells (FIG. 6D). These results suggest CHIR or VPA alone isnot sufficient to maintain the self-renewal of Lgr5⁺ stem cells, butshows synergetic effects when combined with CHIR or other Wntactivators.

In summary, two small molecules, CHIR and VPA, can support Lgr5+ stemcell self-renewal without direct contact with, or in the absence of,Paneth cells. In particular, these small molecules can greatly improvecolony formation from single stem cells, indicating that they provideessential niche signals that are typically provided by Paneth cells.

Example 2 Lgr5⁺ Stem Cells Remain Multipotent Following Culture in CHIRand VPA

Intestinal stem cells have the ability to self-renew as well asdifferentiate into all cell types in the intestine epithelium, includingthe four major cell types: enterocytes, goblet cells, enteroendocrinecells and Paneth cells. To test the differentiation capability of Lgr5⁺stem cells cultured in the CV condition, the cell colonies weretransferred to the ENR condition that permits Lgr5⁺ stem cells tospontaneously differentiate into the mature cell types of the intestine.As expected, after withdrawal of CHIR and VPA, the morphology oforganoids changed to the typical morphology of organoids cultured in ENRcondition, with crypt-villus structure and Lgr5⁺ stem cells at crypttips (FIGS. 7A and 8A). The mRNA expression of differentiation markersAlpi, Muc2, and ChgA elevated and cells expressed a similar level ofLysozyme (comparing ENR and CV in FIG. 7B). Immunocytochemistry stainingfor these markers confirmed the existence of differentiated cell typesin the culture (FIG. 7A).

Example 3 Differentiation of Intestinal Stem Cells is Controlled

Next, with the ability to expand high purity Lgr5+ stem cells in vitro,directing the differentiation of Lgr5⁺ stem cells towards mature celltypes was attempted. As Wnt and Notch are two of the main signalingpathways that control the differentiation of ISC, the Wnt pathwayinhibitor IWP-2 (also I) and Notch inhibitor DAPT (also D) were used toinduce the differentiation of cultured Lgr5⁺ stem cells. Because cellsin the ENR condition spontaneously differentiate into organoidscontaining all epithelial cell types, ENR was included in thedifferentiation cultures. Following culture of single stem cells in theCV condition for 6 days, cell colonies were harvested and transferredinto several wells and cultured in the presence of single or multipleinhibitors (FIG. 8B). As shown in FIG. 7B, replacing CV with IWP-2 orDAPT decreased ISC marker Lgr5 expression and induced expression ofdifferentiation markers Alpi, Muc2, ChgA and Lysozyme. Notably, thepresence of VPA (e.g. comparison between R and V, I and IV, C and CV, orD and DV) caused a lower level of expression of Muc2, ChgA and Lysozymebut not Alpi, indicating VPA specifically suppressed secretory celllineage differentiation. Alternatively, Wnt inhibition with IWP-2preferentially induced Alpi expression, with modestly elevated Muc2 andChgA expression and completely abolished Lysozyme and Lgr5 expression.This indicates that Wnt signaling is required to maintain stemness andto suppress differentiation, yet is also required for Paneth celldifferentiation. The Notch inhibitor DAPT greatly elevated markers ofsecretory cell types including Muc2, ChgA and Lysozyme, which isconsistent with previous reports that Notch inhibition induces secretorycell differentiation (Milano et al., 2004; VanDussen et al., 2012; Wonget al., 2004). Furthermore, the combination of IWP-2 and VPAspecifically induced enterocyte differentiation, presumably by combiningthe effects of both inhibitors, in which IWP-2 induced Lgr5⁺ stem celldifferentiation while VPA suppressed the differentiation of Lgr5⁺ stemcells towards secretory cell types. Similarly, the combination of DAPTand CHIR mainly induced Paneth cell differentiation, and the combinationof IWP-2 and DAPT primarily induced goblet cell differentiation. Theseconditions also induced clear morphological changes which resembled themorphology of each differentiated cell type (FIGS. 7C and 8D). Stainingof enterocyte, goblet cell and Paneth cell markers confirmed the aboveobservations (FIGS. 7C, 7D and 8E, 8F). The presence of IWP-2 or CHIRdid not significantly influence ChgA expression, indicating thatcompared to goblet cells and Paneth cells, the differentiation ofenteroendocrine cells does not strictly require Wnt inhibition oractivation.

Example 4 The Mechanism Mediating the Response of CHIR and VPA isExamined

CHIR is a highly-specific GSK3 inhibitor that activates theWnt/β-catenin signaling pathway (Bain et al., 2007), and has been usedto maintain the self-renewal state of embryonic stem cells (Ying et al.,2008). To confirm that the effect of CHIR was through activating the Wntpathway, the effects of other Wnt pathway activators were tested,including Lithium and Wnt3a. Replacing CHIR with LiCl or Wnt3a increasedcrypt proliferation, indicated by the increased colony size and cellnumbers compared to the ENR condition (FIGS. 9A and 9B). Colonies inthese conditions showed cyst-like structures (FIG. 9A) as shownpreviously (Sato et al., 2011b). Similarly, the effects of other HDACinhibitors including pan-HDAC inhibitors and type-specific inhibitorswere tested. pan-HDAC inhibitor TSA as well as HDAC6 specific inhibitorTubastatin A and Compound 7 showed a similar effect of promoting GFPexpression with VPA (FIGS. 9C and 9D). While other pan-HDAC inhibitorsincluding SBHA and Butyrate, as well as class I (CI-994, MS275, FIG. 9Cand 9D), class IIa (MC1568, FIG. 9C and 9D) and class III (Nicotinamide,FIG. 9F) HDAC inhibitors showed no or only moderate effects to promoteGFP expression (FIG. 9C-9F). TSA and VPA showed marked proliferationinhibition effect at higher concentration, but maintained GFP expressionat both concentrations (FIG. 9E). Of note, Nicotinamide, a SirtuinFamily HDAC inhibitor (class III) that was used in the cultivation ofhuman colon crypts (Jung et al., 2011; Sato et al., 2011a), did notpromote GFP expression or cell proliferation when combined with CHIR orWnt3a (FIG. 9F), indicating it acts through different mechanisms thanVPA. Furthermore, when single Lgr5⁺ stem cells were cultured using CHIRwith TSA or Tubastatin A, or VPA with Wnt3a, BIO or LiCl, cellsexhibited similar colony-forming efficiency, colony morphology and GFPexpression with that of the CV condition (FIG. 10).

Previous reports have shown that Notch pathway activation is required toinhibit secretory cell differentiation and maintain self-renewal of stemcells, which is consistent with the effects of VPA treatment. WhetherVPA targets elements of the Notch pathway to exert its effects wasevaluated. First, rescue of Notch inhibition by the addition of VPA wastested. Treatment with y-secretase inhibitor DAPT led to impaired cellproliferation and GFP expression, which was rescued by VPA in a dosedependent manner (FIG. 11A). This suggests VPA acts downstream of NICDformation and could bypass the requirement of ligand-receptor mediatedNotch activation.

VPA was previously shown to activate the Notch pathway in cancer cellslines (Greenblatt et al., 2007; Stockhausen et al., 2005). Toinvestigate the effect of VPA on the activation of the Notch pathway,cells cultured in ENR or ENRC conditions were treated with VPA andanalyzed for the expression of Notch pathway genes. It was determined,however, that addition of VPA to ENR or ENR-C for 24 hours moderatelydecreased the expression of Notch1 or Hes1, which is a downstream targetgene of Notch (FIGS. 11B and 11C). Additionally, a pronounced decreaseof the negative Notch target Atoh1 (Math1) in cells treated with VPA andCHIR for 24 hours or 6 days was observed (FIG. 11B-11D). Atoh1 has beenshown to be essential for the differentiation of ISC towards secretorycell lineage (van Es et al., 2010; Yang et al., 2001). Intestinal stemcells remain functional both in vivo and in vitro after Paneth cellablation induced by Atoh1 deficiency (Durand et al., 2012; Kim et al.,2012). Atoh1 inhibition after CHIR or CHIR+VPA treatment would helpmaintain the self-renewal program of intestinal stem cells.

Accordingly, the control of the self-renewal of Lgr5⁺ intestinal stemcells and their differentiation towards differentiated cell types in theintestinal epithelium in vitro has now been achieved through using acombination of growth factors and small molecule inhibitors, whichclosely mimics the in vivo intestinal epithelial biology (FIGS. 12A and12B). Under physiological conditions (FIG. 12A), the self-renewal anddifferentiation of ISCs are controlled by the cooperation of Wnt andNotch pathways. The activation of both pathways (indicated by Wnt On,and Notch On) maintains ISCs in an undifferentiated, self-renewingstatus. The deactivation of the Notch pathway (Notch Off) leads to thespecification of secretory cell types and further deactivation of theWnt pathway (Wnt Off) leads to goblet cell differentiation. Continuousactivation of the Wnt pathway in the absence of Notch leads to Panethcell differentiation. There is no strong dependence of the Wnt pathwayfor enteroendocrine cell differentiation. Alternatively, continuousNotch activation and Wnt deactivation leads to enterocyte celldifferentiation. When Lgr5+ stem cells are cultured in vitro (FIG. 12B),CHIR99021 activates the Wnt pathway and inhibits enterocytedifferentiation while VPA alone or together with CHIR suppressessecretory cell specification. The combination of CHIR and VPA maintainsISCs in an undifferentiated, self-renewing status. The inhibition of theNotch pathway with DAPT leads to the specification of secretory celltypes and further addition of CHIR leads to Paneth cell differentiation,while addition of the Wnt pathway inhibitor IWP-2 leads to goblet celldifferentiation. Alternatively, the combination of IWP-2 and VPA, whichinduces differentiation and suppresses secretory cell specificationleads to enterocyte differentiation.

Example 5 Proliferation of Lgr5-Positive Stem Cells Derived from InnerEar is Increased in the Presence of CHIR and VPA

Sensory hair cells of the mammalian organ of Corti in the inner ear donot regenerate upon damage. Li et. al., 2003, found that adult utricularsensory epithelium contains cells that display the characteristicfeatures of stem cells. These inner ear stem cells can be cultured invitro as suspension spheres in the presence of EGF, bFGF and IGF-1 (Liet al., 2003). Later, it was found that post-mitotic supporting cellsretain the ability to divide and trans-differentiate into new hair cellsin culture (Patricia et al., 2006, Nature), suggesting these supportingcells may be inner ear stem cells. Purified cochlear supporting cellscan be cultured in vitro in the presence of EGF, bFGF on embryonicperiotic mesenchymal feeder cells. (Patricia et al., 2006). Shi et alfound that a subset of supporting cells in the newborn and adult murinecochlea express Lgr5, a marker for adult stem cells (Shi et al., 2012).Importantly, Lgr5-positive cells can be isolated and cultured in asingle-cell suspension, in the presence of EGF, bFGF and IGF-1, anddisplay enhanced self-renewal capacity compared with Lgr5-negativecells. Previous inner ear stem cell cultures utilized a suspensionculture method in which only approximately 0.068% of total cells (Li etal., 2003) or 2% of sorted Lgr5-positive cells could form spheres (Shiet al., 2012), probably because of inadequate growth environment for thecells. As described herein, a highly efficient in vitro culture systemfor inner ear stem cells has now been developed.

Isolated mouse cochlea from P1 to P2 Lgr5-GFP mice containedLgr5-positive cells as shown in FIG. 13A. The same culture condition(EGF, Noggin, R-spondin1, or “ENR”) as used in Lgr5+ small intestinalstem cell cultures was first established. As shown in FIG. 13B, thecombination of EGF, Noggin and R-spondin1 increased the colony-formingefficiency from single cochlear epithelial stem cells compared to EGFalone. As expected, the combination of CHIR and VPA, but not CHIR alone,greatly increased the colony-forming efficiency, cell proliferation andGFP expression of the cells. Surprisingly, removing Noggin from theENR-CV combination (the “ER-CV” condition) resulted in slightly highercolony-forming efficiency and higher GFP expression level, as shown bybrightfield and GFP images in FIG. 13B. These results indicate that Wntpathway activation by R-spondin1 or CHIR promotes the proliferation ofinner ear stem cells and the combination of CHIR and VPA greatlypromotes the proliferation and self-renewal of inner ear stem cells.

Mitogenic growth factors including EGF, bFGF and IGF-1 were previouslyused in the suspension culture system and shown to promote sphereformation of isolated inner ear stem cells (Li et al., 2003; Shi et al.,2011). Next the effects of CHIR and VPA were tested in the presence ofthese growth factors as described in Table 1.

TABLE 4 Cell Culture Solutions Stock Final Reagent Name Supplier Cat#Solution Concentration Control Advanced DMEM/F12 Invitrogen 12634-Medium 010 GlutaMAX Invitrogen 35050- 200 mM 2 mM 061Penicillin/Streptomycin Invitrogen 15140- 10000/10000 U/ml 122 100/100U/ml HEPES Invitrogen 15630- 1M 10 mM 080 N2 Supplement Invitrogen17502- 100x 1x 048 B27 Supplement Invitrogen 12587- 50x 1x 010N-Acetylcysteine Sigma- A9165 500 mM 1 mM Aldrich Growth EGF RecombinantInvitrogen PHG0311 500 μg/ml 50 ng/ml factors Human Protein MouseRecombinant Peprotech 250-38 100 μg/ml 100 ng/ml Noggin HumanRecombinant Peprotech 120-38 500 μg/ml 50 ng/ml R-Spondin 1 HumanRecombinant Invitrogen PHG0024 100 μg/ml 10 ng/ml FGF-Basic IGF-1Recombinant Invitrogen PMG0078 100 μg/ml 50 ng/ml Human protein SmallCHIR-99021 LC C-6556 10 mM 3 μM molecules Laboratories Valproic acidsodium Sigma- P4543 1M 1 mM salt Aldrich Y-27632 Sigma- Y0503 10 mM 10μM Aldrich Trichostatin A Sigma- T8552 10 mM 20 nM Aldrich Sodiumbutyrate Sigma- B5887 1M 0.5 mM Aldrich

Isolated organ of Corti from Lgr5-GFP mice were dissociated into singlecells using accutase and cultured in multiple combinations of solublefactors and small molecules in Matrigel for 8 days. The resultingcultures were further dissociated into single cells and analyzed usingFACS. Consistent with previous results, the addition of CHIR and VPA,but not CHIR or VPA alone, greatly increased cell proliferation (9-20fold) and GFP expression as shown by the percentage of GFP+ cells (60fold) and relative GFP intensity of GFP+ cells (2 fold) (FIG. 14A and14B). In addition, the combination of EGF, bFGF and IGF-1 (designated asEFI) improved cell proliferation and GFP expression compared with theENR condition (FIG. 14A-14C).

To further investigate the effects of individual growth factors whencombined with CHIR and VPA, growth factors including Mitogenic growthfactors (EGF, bFGF and IGF-1) as well as the Wnt agonist R-spondin 1 incombination with CHIR and VPA were tested. The addition of EGF to the CVcondition greatly increased cell proliferation as indicated by increasedcell number in the culture. Addition of bFGF but not IGF-1 or R-spondin1 to EGF+CV further increased cell proliferation and GFP expression(FIG. 14D). Although the addition of IGF-1 or R-Spondin 1 to theEGF+bFGF combination slightly increased GFP expression (FIG. 14E), wefound that they are not essential to maintain the proliferation and GFPexpression of cultured cells (FIG. 14F).

Example 6 Lgr5-Positive Intestinal Stem Cells Form Transplantable Crypts

To examine the potential to transplant intestinal stem cells, theengraftment of small intestinal crypts was tested on healthy colontissue in vitro. Colon tissue was harvested from wild type mice andopened longitudinally. A 1 cm fragment was removed and washed with PBS.The epithelial layer was removed by scraping using a surgical blade andthe tissue was placed into a 24 well plate. Small intestinal cryptsisolated from Lgr5-GFP mice were stained with a DiD membrane dye andplaced onto the colon tissue within 5-10 μl of crypt culture mediacontaining advanced DMEM/F12 (Invitrogen), 2 mM GlutaMax (Invitrogen),10 mM Hepes (Invitrogen),100 U/ml Penicillin/100 ug/ml Streptomycin(Invitrogen), 1× N2 supplement (Invitrogen), 1× B27 supplement(Invitrogen), 50 ng/ml EGF (Peprotech), 500 ng/ml R-spondin 1 (R&DSystems), 10 μM Y-27632 (Rho Kinase inhibitor, Sigma-Aldrich; and 100ng/ml Noggin (Peprotech). The tissue was further incubated at 37° C. for30-60 minutes in a humidified environment to permit the adherence ofcrypts. Crypt culture media were then added into the wells and thecrypts were further cultured for 7 days. Seeded crypts attached to thecolon and spread in 24 hours (FIG. 15). Fluorescent image showed cryptsengrafted on the colon in 48 hours (FIG. 16) and maintained Lgr5-GFPexpression for at least one week (FIG. 17).

To further test the engraftment ability of small intestinal crypts, aTRUC mouse model that exhibits spontaneous Ulcerative Colitis and mimicsthe human condition was used. Prolapsed tissue was excised from the TRUCmouse and washed with PBS and placed into a 24 well plate. Smallintestinal crypts were stained with DiD and placed onto the prolapsetissue. The tissue was then incubated at 37° C. for 30-60 min in ahumidified environment to permit the adherence of crypts. Crypt culturemedia was added into the wells. The prolapsed tissue and crypts werefurther cultured in vitro for 2 days. As expected, crypts engrafted onthe prolapsed tissue (FIG. 17).

Example 7 Patch Culture Systems for Small Intestine Organoids Mimic the3-Dimensional Physiological Environment

An in vitro culture system capable of supporting the growth oflarge-scale, organized 3-dimensional cellular structures (e.g.organoids) on a submucosal scaffold has now been developed. As describedbelow, an improved small intestinal submucosa (“SIS”)-based culturesystem for 3-dimensional tissue constructs was prepared by seeding thesubmucosa with a preselected cell type and facilitating growth with aunique collagen-based overlay. This overlay, initially a viscous fluidpre-polymerization, is used to coat seeded, early stage cells ororganoids (subcultured from cells), as well as to coat the SIS base toencase the cells in a collagen residue (FIG. 19E and 19F). Afterpolymerization, the liquid solidifies to maintain its positioncontacting cell membranes as well as SIS and promotes organoidexpansion. It has now been discovered that varying the composition ofSIS with this overlay facilitates cell adhesion and growth. This willfacilitate in vitro, as opposed to in vivo, tissue maturation. This is aunique improvement over other submucosal-based and similar syntheticsystems in that 3-dimensional expansion of adhered cells into large,endogenous-type organoids is achieved prior to transplantation.

Additionally, a method to support 3-dimensional organoid growth onsubmucosa at rates comparable to Matrigel without the use of gel layershas also been discovered. This system is composed of vertebrate SIS andpreselected cells, seeded on the SIS patch. Preselected bioactive agentsare infused in the patch prior to cell seeding to support this gel-freeculture system (FIG. 19C and 19D).

To develop the patch culture system, varying combinations of an SIS baseand collagen overlay with infused growth factors (FIG. 19E and 19F) wereexplored. This permitted the creation of a more physiological tissueinterface with a transition from stiff (SIS) to soft (collagen) matrix.It was determined that seeded cells and organoids coated with a collagenresidue are provided with a 3-dimensional environment similar to thatprovided by Matrigel. As such, this system is a suitable replacement forMatrigel in culturing 3-dimensional organoid constructs. The majority ofseeded cells or organoids are both adhered to the SIS on the lower halfof the cell membranes but also enveloped by polymerized collagen onnon-adhered regions of the membrane (FIG. 19E, inset). Thus, each cellmembrane is functionally encased in a form of matrix, whether it is SISor collagen. In some samples, a variety bioactive agents was employed tosupport cell and organoid seeding, growth, and differentiation beyondSIS alone (FIG. 19F). While the applicants describe an infusion ofbiomolecules specific to intestinal stem cell culture, it is declaredthat biomolecules can be tailored to aid in the growth of other seededcells from different tissues including pancreas, breast, liver, andstomach tissues. Accordingly, tissues-specific biomolecules may selectedfrom the following: antiviral agent, antimicrobial agent, antibioticagent, amino acid, peptide, protein, glycoprotein, lipoprotein,antibody, steroidal compound, antibiotic, antimycotic, cytokine,vitamin, carbohydrate, lipid, extracellular matrix, extracellular matrixcomponent, chemotherapeutic agent, cytotoxic agent, growth factor,anti-rejection agent, analgesic, anti-inflammatory agent, viral vector,protein synthesis co-factor, hormone, endocrine tissue, synthesizer,enzyme, polymer-cell scaffolding agent with parenchymal cells,angiogenic drug, small molecule, nanoparticle, collagen lattice,antigenic agent, cytoskeletal agent, nucleic acid, cellular attractant.

To begin, crypts were isolated in accordance with previous methods (Satoet al., 2009, Yui et al., 2012). Murine small intestine was isolated,incised longitudinally and washed in ice-cold PBS to clear luminalcontents. Fragments were cut into 2 mm pieces, transferred to a 50 mlfalcon tube and gently washed in 50 ml of ice-cold PBS using a 10 mlpipette. The supernatant was removed and the process was continued untilthe supernatant cleared. Fragments were incubated for 45 minutes at 4°C. in PBS containing 2 mM EDTA to release crypts. The supernatant wasremoved and fragments pipetted up and down with 50 ml of PBS. Once thesupernatant was confirmed to contain the crypt fraction, the suspensionwas filtered through a 70 μm cell strainer and spun in a centrifuge at300 g for 5 minutes. Crypts were re-suspended in 10 ml of ice-cold basalculture media (containing advanced DMEM/F12 (Invitrogen) 2 mM GlutaMax(Invitrogen), 10mM Hepes (Invitrogen) and 100 U/ml Penicillin/100 ug/mlStreptomycin (Invitrogen)) and transferred to a 15 ml falcon tube. ThePBS wash was repeated and the crypts were spun at 200 g for 2 minutes toremove single cells. Crypts were counted and plated in a 48 well platewith either Matrigel or Collagen I (consisting of 100 ul 10× PBS, 4.9 μlNaOH, 684 μl H20 and 211 μl collagen type I (rat tail high concentration9.49 mg/ml; BD Biosciences) at a concentration of 1000 crypts per well,each well containing 200 μl of matrix. After polymerization of thechosen gel product, 500 μl of 1× standard crypt culture medium (serumfree) was added, containing advanced DMEM/F12 (Invitrogen), 2 mMGlutaMax (Invitrogen), 10 mM Hepes (Invitrogen),100 U/ml Penicillin/100ug/ml Streptomycin (Invitrogen), 1× N2 supplement (Invitrogen), 1× B27supplement (Invitrogen), 50 ng/ml EGF (Peprotech), 500 ng/ml R-spondin 1(R&D Systems), 10 μM Y-27632 (Rho Kinase inhibitor, Sigma-Aldrich; and100 ng/ml Noggin (Peprotech). Cells were grown for 4-5 days beforeseeding onto the patch, changing media every other day. Y-27632 was onlyincluded in the culture media for the first 48 hours.

After 4-5 days in culture the Lgr5+ organoids were passaged using amodified protocol described previously (Sato et al., 2009). Culturemedia was removed from the matrigel, which was then manually broken witha p1000 pipette and then transferred to a BSA coated 15 ml falcon tube.Collagen gels were incubated in DMEM containing collagenase type XI at37° C. for 5 minutes and then transferred to a BSA coated 15 ml falcontube. Basal media was added and organoids were gently disrupted withfrequent inspection by inverted microscopy until the majority oforganoids were single-crypts. Organoids were washed in 10 ml of basalmedia and centrifuged at 200 g for 2 minutes. The pellet was resuspendedin crypt culture media at a concentration of 500 single-crypt organoidsper 500 μl.

Patches were generated and prepared for seeding inside the wells of astandard 48-well plate (one patch per well, luminal side up). SIS wascut into the desired length to cover the bottom of each well (˜1 cm for48 well plate). Isolation of SIS has been previously described (Badylaket al., 1989). Using blunted forceps, each SIS segment was transferredto the bottom of a well and carefully spread to its full diameter,luminal side facing up. Orientation was confirmed by analysis underinverted microscopy to visualize the acellular remnants of crypts on thesuperficial surface. Depending on the compliance and strength required,multiple layers of SIS can be layered and bonded together. In this case,each segment can be spread on top of one another for the desired numberof segments and the patch gently compressed with forceps and allowed toair dry at 5% CO₂, 37° C. for 5 minutes. Prior to seeding, each patchsegment was dehydrated by passive evaporation for 24 hours and infusedwith concentrated crypt culture media and optionally, small molecules asdescribed below. Specifically, each segment of the patch was placed andspread, luminal side up, in the well of a 48 well plate, and 100 μl ofconcentrated factors (EGF, Noggin, R-spondin 1, Y-27632, Valproic Acidand CHIR) was deposited for 24 hour incubation at 5% CO₂, 37° C.

Individual 500 μl single-crypt organoid samples were deposited into awell containing a patch base and incubated for 24 hours at 5% CO₂ and37° C. (FIG. 20A). The seeded patches were maintained in culture mediafor 24 hours to allow for firm adherence and to obtain nutritionalsupport from embedded growth factors in the patch (FIG. 20B).

In some samples, a thin collagen gel residue (termed as the gel-patch)was coated onto the top of the patch/organoid complex to provide aminimal but functional 3-dimensional environment for each organoid.Physical and chemical cues obtained from the cell surface enhance3-dimensional cell structure proliferation in order to replicatephysiological morphology (Seidi, A., et al., 2011). Collagen I matrix(20-40 μl) was layered on seeded patches, taking care to leveragesurface tension to prevent spreading of the gel beyond the patch (FIG.20C) and the well plate incubated at 5% CO₂, 37° C. for 30 minutes.Crypt culture media (500 μl) was deposited into each well and changedevery other day.

In some samples, the patch was incubated in growth factors prior toseeding to examine whether it would facilitate adherence of organoids inthe first 24 hours. Accordingly GF-infused (including EGF, Noggin,R-spondin 1, Y-27632, Valproic Acid and CHIR) versus non-infused patches(SIS in PBS) were seeded. In this assay, non-infused patches used basalmedia in lieu of culture media to deprive organoids of media growthfactors as well.

Growth of intestinal organoids was assessed by quantifying the number ofcrypts per organoid in 7 separate systems: Matrigel (control), thegel-patch system with infused Growth Factors (referred to herein as GFs,and including EGF, Noggin, R-spondin 1, Y-27632, Valproic Acid andCHIR), the bare patch with infused GFs but without collagen overlay,Collagen I gel only, Collagen I gel with GFs added to directly to theculture media (including EGF, Noggin, R-spondin 1, Y-27632, ValproicAcid and CHIR), Collagen I gel with GFs embedded in the gel itself(including EGF, Noggin, R-spondin 1, Y-27632, Valproic Acid and CHIR),and the bare patch without collagen overlay or infused GFs. Other thanthe Collagen I group with GFs and small molecules added directly to themedia, all culture media was standard between each system, was changedevery other day, and included EGF, Noggin, R-spondin 1, Y-27632 (first48 hours only). Standard crypt culture media is described above.

The experiment was conducted over 96 hours and daily quantification oforganoid growth was documented by visually inspecting the number ofcrypts per organoid. The gel-patch system with GFs was able to supportorganoid growth at levels comparable to Matrigel controls (FIG. 19). Thebare patch, without GFs, was not able to support measurable organoidgrowth. Upon closer inspection, the bare SIS patch appeared to growLgr5+ cells in sheets as opposed to 3-dimensional organoids. However,the bare patch with infused GFs (EGF, Noggin, R-spondin 1, Y-27632,Valproic Acid and CHIR) supported organoid growth on par with both thegel-patch system and Matrigel. This indicates that, with sufficient GFsupport, a gel-free culture system is capable of sustaining short-term,3-dimensional organoid growth on par with Matrigel. While Collagen Ialone facilitates moderate organoid growth, SIS infused with GFs is aviable replacement for collagen's 3-dimensional growth promoting effect.Furthermore, when the same GFs (EGF, Noggin, R-spondin 1, Y-27632,Valproic Acid and CHIR) were added directly to the culture media of theCollagen I gel culture, organoid growth rates remained low.Additionally, when Collagen I gel was prepared with the aforementionedGFs embedded directly in gel prior to seeding, organoid growth ratesremained low. GFP signal was maintained throughout the gel-patch system(representative example in FIG. 21B and 21C). The observation that thebare patch (SIS without a collagen overlay or GFs) failed to supportstructured organoid growth reaffirms the importance of sufficientphysical and chemical cues to promote 3-dimensional structures.

SIS or collagen alone has been used in the literature as a base scaffoldfor cell seeding, resulting in the formation of cellular monolayers(Baumert et al. 2007; Campodonico et al. 2004; Feil, G., et al. 2006;Zhang, Y., et al. 2000). By contrast, growing cells at the interface ofthese two matrices favors 3-dimensional organoid growth over monolayergrowth. This mimics the physiological environment more closely, allowingfor accelerated and structured growth. Importantly, these resultsdescribe a patch culture system for small intestine organoids that is asuperior alternative to Matrigel. Matrigel-based transplantation inanimal models has encountered significant barriers in moving towards ahuman model, the most critical including biocompatibility issues.Growing a 3-dimensional cell-based structure often requires embedding athick matrix gel. The patch culture system overcomes this requirementwhile providing comparable results. Replacing Matrigel with acombination of endogenous extracellular matrix materials and specificbioactive growth factors avoids biocompatibility issues whilemaintaining 3-dimensional organoid, ex vivo growth. A timelapse image of3-dimensional, ex vivo organoid expansion from an initial seed isdemonstrated in FIG. 22.

Whether incubation of the patch in growth factors prior to seedingfacilitates adherence of organoids in the first 24 hours was evaluated.The seeding efficiency was compared in growth factor infused patches(including EGF, Noggin, R-spondin 1, Y-27632, Valproic Acid and CHIR)versus non-treated patches (stored in PBS). The assay was conducted bymeasuring the percentage of organoids retained after media washes at 4and 12 hours when cells are cultured solely in media devoid of growthfactors (basal media only). When SIS was omitted and organoids wereseeded directly on plastic collagen-coated and non-collagen coatedwells, dissociation of all organoids occurred within 24 hours. However,SIS patches maintained the majority of organoids at 24 hours, in bothstructure and GFP expression. An improvement in adherence was observedwhen cells were seeded on growth factor infused patches (including EGF,Noggin, R-spondin 1, Y-27632, Valproic Acid and CHIR). Therefore, growthfactor infusion may also be useful to provide adequate nutrition andfactors during culture and following transplantation while bridging thegap to cellular engraftment.

Example 8 Implanted Patch Exhibits Growth-Promoting Properties In Vivo

Acellular, gel-free variations of the patch system were tested toevaluate mucosal healing properties in vivo. A rat surgical model ofmucosal defects was designed in order to test the growth-promotingproperties of the implantation patch in vivo. The implantation patch wasassembled by carefully spreading a portion of SIS, luminal side facingup, over 6 mm, circular poly(glycerol sebacate) urethane (PGSU)backings. The patch was incubated at 5% CO₂, 37° C. for 30 minutes toallow bonding of the PGSU and SIS. A 4 mm defect was created in thegastric wall via punch biopsy, as shown in FIG. 23. Acellular patches (6mm in diameter) were placed over the external gastric wall, carefullycovering the defect with the chosen material. The patch was secured byan adapted Graham patch method (So, et al., 1996) using sutures andnearby connective tissue. Three variants of acellular patches wereapplied, including a) PGSU-backed SIS patch (without GFs), b)PGSU-backed SIS patch with GFs infused (EGF, Noggin, R-spondin 1,Y-27632, Valproic Acid and CHIR) and c) PGSU backing only (no SIS). Atno point was peritonitis observed in any rat. One week afterimplantation over mechanically-induced gastric wall defects, the gastrictissue containing the defect and patch implants was harvested to conducthistological examination of tissue.

It was hypothesized that implanting variations of the patch would showvarying degrees of mucosal healing. Gross examination demonstrated asignificant benefit in the SIS patch with GFs, as the defect wasepithelialized and closed. Partial closure without epithelialization wasobserved in SIS patches without GFs, and no closure or epithelializationwas observed in the PGSU only (control) patches. Histologicalexamination revealed mild inflammation in both the SIS patch with andwithout GFs, but no stomach content leakage. Histological examination ofthe PGSU-only patch demonstrated moderate inflammation as well as thepresence of Giant cells, likely responding to leakage of stomachcontents. Accordingly, patch culture systems described herein aretransplantable from the culture dish directly to the patient, withincreased translational potential, as the patch is rigid and less likelyto obstruct in small spaces (e.g. the intestinal lumen, vascular spaces)given its low height profile.

Example 9 Culturing of Human Small Intestinal Crypts/Stem Cells

Human small intestinal crypts were isolated from a resected normal smallintestinal specimen and cultured as described in Example 1. The samecell culture solutions used in the culture of mouse small intestinalstem cells/crypts, which comprise a combination of CHIR99021 and VPA orTubastatin A added to the ENR (EGF, Noggin, R-Spondin 1) condition, werecompared to published cell culture solutions for human intestinal stemcells/crypts (Jung et al., 2011; Sato et al., 2011). RT-PCR was used toassess the maintenance of epithelial stem cells in the culture,specifically by determining the self-renewal or differentiation status.LGR5 was used as a stem cell marker and ALPI, MUC2, CHGA and LYZ wereused as differentiation markers. Cell growth was assessed by countingthe cell number in the cultures and by observing the morphology and sizeof colonies.

Similar to the mouse intestinal stem cell culture, the combination ofCHIR+VPA or CHIR+Tubastatin A greatly promoted the expression of stemcell marker LGR5, suggesting the cultured cells were enriched in stemcells (FIG. 24). Notably, the culture condition containing CHIR and VPAor CHIR and Tubastatin A outperformed published conditions in promotingLGR5 expression (FIG. 24). In addition, individual components showingimprovement to the culture media were tested, including A83-01(ALK4,5,7, Tgf-β inhibitor), SB202190 (p38 inhibitor) and Nicotinamide(Vitamin B derivative). It was determined that 10 mM Nicotinamideincreased the proliferation of human small intestinal crypts when addedto the CHIR+VPA condition, as indicated by the increased cell number inthe culture (FIG. 25A), without great impact on LGR5 expression (FIG.25B). While the combination of A83-01 and SB202190 (AS) increased theproliferation of cells (FIG. 25A), they greatly decreased the expressionof LGR5 (FIG. 25B). In addition, a lower concentration of VPA (0.5 mM,compared to that used in mouse cultures (1-2 mM)) increased cellproliferation of human small intestinal crypts (FIG. 25A). Collectively,it was determined that the culture condition containing EGF, Noggin,R-spondin1, CHIR, VPA (0.5 mM) and Nicotinamide or EX527 was an optimalculture condition for human intestinal stem cells. In this condition,isolated small intestinal crypts grow into colonies comparable to mousesmall intestinal stem cells (FIG. 26).

Example 10

To test the in vivo effect of CHIR and VPA on intestinal epithelialcells, CHIR99021 (30 mg/Kg in 100 μl DMSO) and VPA (200 mg/Kg in 100 μlwater) were administered to 4-6 week old female Lgr5-GFP mice viagavage. Control mice were given a mixture of 100 μl DMSO and 100 μlwater. Drugs were administered every 48 hours for 7 days (at Day 0, Day2, Day 4 and Day 6). At Day 7, mice were sacrificed and intestine tissuewere collected. The small intestine were further washed with PBS, fixedwith 4% PFA for 12 hours, embedded in Paraffin and stained usingstandard Hematoxylin and eosin (H&E) staining protocol. Images wereacquired using an inverted microscope (EVOS, Advanced Microscopy Group).In vivo administration of CHIR and VPA increased the size of cryptsfollowing 3 administrations over the course of 7 days (FIG. 27).

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Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. A method of increasing leucine-rich repeat-containing G-proteincoupled receptor 5 (LGR5) expression or numbers of LGR5 positiveepithelial cells within epithelial tissue in a subject in need thereof,the method comprising administering CHIR99021 and valproic acid sodiumsalt to the subject in an amount sufficient to increase LGR5 expressionor numbers of LGR5 positive epithelial cells within epithelial tissue,wherein the epithelial cells are present within an inner ear of thesubject, thereby increasing LGR5 expression or numbers of LGR5 positiveepithelial cells within the epithelial tissue in the subject.
 2. Themethod of claim 1, wherein sensory hair cells of the organ of Corti inthe inner ear of the subject are damaged.
 3. The method of claim 1,wherein the method increases proliferation of inner ear stem cells inthe subject.
 4. The method of claim 1, wherein the subject is human. 5.The method of claim 1, wherein CHIR99021 and valproic acid sodium saltare administered concomitantly.
 6. The method of claim 5, whereCHIR99021 and valproic acid sodium salt are administered as a singlecomposition.
 5. hod of claim 5, wherein CHIR99021 and valproic acidsodium salt are administered as separate compositions.
 8. The method ofclaim 1, wherein CHIR99021 and acid sodium salt are administeredsequentially.